Plasma carboxypeptidase

ABSTRACT

A novel polypeptide, designated plasma carboxypeptidase B (PCPB), has been purified from human plasma. It has been cloned from a human liver cDNA library using PCR amplification. Provided herein is nucleic acid encoding PCPB useful in diagnostics and in the recombinant preparation of PCPB. PCPB is used in the preparation and purification of antibodies thereto, in the purification of plasminogen, in the inhibition of plasminogen activation by t-PA in the presence of fibrinogen, and in diagnostic assays.

This is a divisional application of U.S. patent application Ser. No.08/277,540 filed on Jul. 19, 1994, now U.S. Pat. No. 5,474,901, which isa divisional of 08/167,727 filed Dec. 15, 1993, now U.S. Pat. No.5,364,934, which is a continuation of U.S. patent application Ser. No.07/959,944 filed Oct. 14, 1992, now abandoned, which is a divisional ofU.S. patent application Ser. No. 07/649,591 filed Feb. 1, 1991, now U.S.Pat. No. 5,206,161.

FIELD OF THE INVENTION

This application relates to a carboxypeptidase that binds plasminogen.In particular, it relates to a plasma carboxypeptidase designated plasmacarboxypeptidase B (PCPB) sharing some sequence identity withcarboxypeptidase A and rat pancreas carboxypeptidase B that inhibits theenzymatic conversion by tissue plasminogen activator of plasminogen toplasmin in the presence of fibrinogen and does not inhibit plasminactivity.

BACKGROUND OF THE INVENTION

The carboxypeptidase family of exopeptidases constitute a diverse groupof enzymes that hydrolyze carboxyl-terminal amide bonds in polypeptides.Carboxypeptidases from grains such as wheat and barley have beenisolated and sequenced (Baulcombe et al., J. Biol. Chem., 262:13726-13735 [1987]; Svendsen and Breddam, Carlsberg Res. Commun., 52:285-295 [1987]), as well as carboxypeptidases from bacteria, and acarboxypeptidase Y has been isolated from yeast vacuoles and sequenced.Valls et al., Cell, 48: 887-897 [1987]; Svendsen et al., Carlsberg Res.Commun., 47: 15-27 [1982]). The sequences of carboxypeptidase B fromcrayfish (Titani et al., Biochemistry, 23: 1245-1250 [1984]) and Africanlungfish (Reeck and Neurath, Biochemistry, 11: 3947-3955 [1972]) havebeen determined. A large number of mammalian tissues also produce theseenzymes.

The exocrine pancreas synthesizes and secretes a subset of zincmetalloproteases. Two members of this metalloprotease family,carboxypeptidase A and carboxypeptidase B, have been purified frombovine pancreas and characterized. Barrett and MacDonald, MammalianProteases, a Glossary and Bibliography, Vol. 2 (Academic Press, London1985), and references cited therein, including Titani et al., Proc. Nat.Acad. Sci. USA, 72: 1666-1670 [1975]; Bradshaw et al., Biochemistry, 10:961-972 [1971]; Wade et al., Biochimie, 70: 1137-1142 [1988]. Bovinecarboxypeptidase B was found to inhibit the activation of plasminogen byt-PA in the presence of degraded fibrin. Pannell et al., J. Clin. Inv.,81: 853-859 (1988).

The bovine carboxypeptidases A and B have similar amino acid sequence,three-dimensional structure, and catalytic mechanisms, but differ in thesubstrate upon which they act. Carboxypeptidase A hydrolyzescarboxyl-terminal amide bonds in which the adjoining carboxy-terminalamino acid contains an aromatic or branched aliphatic side chain,whereas carboxypeptidase B prefers Lys or Arg residues as substrates atthe carboxyl terminus.

Three carboxypeptidases differing in their substrate specificity(designated CPA1, CPA2, and CPB) were isolated from rat pancreas.Gardell et al., J. Biol. Chem., 263: 17828-17836 (1988). The genes forrat CPA1 and CPA2 have been cloned and sequenced. Gardell et al., supra;Clauser et al., J. Biol. Chem., 263: 17837-17845 (1988). See also Quintoet al., Proc. Natl. Acad. Sci. USA, 79: 31-35 (1982). Sequences havealso been obtained of fragments from porcine carboxypeptidase A(Vendrell et al., Biochem. Biophys. Res. Commun., 141: 517-523 [1986])and carboxypeptidase B (Aviles et al., Biochem. Biophys. Res. Commun.,130: 97-103 [1985]).

In addition to the pancreas, mast cells also contain large amounts ofcarboxypeptidase A, including rat and mouse peritoneal connective tissuemast cells [Everitt and Neurath, FEBS Lett., 110: 292-296 (1980);Schwartz et al., J. Immunol., 128: 1128-1133 (1982); Serafin et al., J.Immunol., 139: 3771-3776 (1987)], mouse Kirsten sarcomavirus-immortalized mast cells [Reynolds et al., J. Biol. Chem., 263:12783-12791 (1988)], and human skin mast cells [Goldstein et al., J.Immunol., 139: 2724-2729 (1987); Goldstein et al., J. Clin. Invest., 83:1630-1636 (1989)].

Mast cell carboxypeptidase A is a neutral to basically charged proteinstored in the secretory granules of rat and mouse peritoneal connectivetissue mast cells and mouse interleukin-3-dependent bone marrow-derivedmast cells as a fully active enzyme bound ionically to acidicallycharged proteoglycans. Basically charged serine endopeptidases aresimilarly stored. The close approximation of carboxypeptidase A andserine protease activities within the protease-proteoglycanmacromolecular complex is thought to facilitate sequential endopeptidaseand exopeptidase cleavages of common protein substrates. Kokkonen andKovanen, J. Biol. Chem., 264: 10749-10755 (1989); Kokkonen et al., J.Biol. Chem., 261: 16067-16072 (1986).

Mast cell carboxypeptidase A has been isolated from the secretorygranules of mouse peritoneal connective tissue mast cells and from amouse Kirsten sarcoma virus-immortalized mast cell line, and a cDNAencoding this exopeptidase has been cloned. Reynolds et al., J. Biol.Chem, 264: 20094-20099 (1989). In addition, the mast cellcarboxypeptidase A from humans has been cloned and its sequencedetermined. Reynolds et al., Proc. Natl. Acad. Sci. USA, 86: 9480-9484(1989).

Other mammalian carboxypeptidases besides carboxypeptidase B thatspecifically remove terminal basic amino acids include carboxypeptidaseH (also known as enkephalin convertase or carboxypeptidase E),carboxypeptidase M, and carboxypeptidase N. The mammalianarginine/lysine carboxypeptidases have important functions in manybiological processes, including protein digestion, activation,inactivation, or modulation of peptide hormone activity, and alterationof the physical properties of proteins and enzymes.

The actual role of these carboxypeptidases in vivo is likely related totheir localization as well as their physical properties. For example,pancreatic carboxypeptidase B is not normally found outside the pancreasor small intestine except in the case of acute pancreatitis, consistentwith its major function in protein degradation in the digestive tract.Delk et al., Clin. Chem., 31: 1294-1300 (1985).

In contrast, human plasma carboxypeptidase N circulates in plasma as alarge tetrameric complex of two active subunits (48-55 kD) and twoglycosylated inactive subunits (83 kD) that stabilize the activesubunits and keep them in the circulation. Carboxypeptidase N protectsthe body from potent vasoactive and inflammatory peptides containingCOOH-terminal Arg or Lys released into the circulation. Erdos (ed.) inHandbook of Experimental Pharmacology, Vol. 25, Supplement, pp. 428-487,(Springer-Verlag, Heidelberg, 1979); Plummer and Hurwitz, J. Biol.Chem., 253: 39-7-3912 (1978). Recently, carboxypeptidase N has beencloned and sequenced. Tan et al., J. Biol. Chem., 265: 13-19 (1990);Gebhard et al., Eur. J. Biochem., 178: 603-607 (1989); Skidgel et al.,Biochem. Biophys. Res. Commun., 154: 1323-1329 (1988).

Carboxypeptidase E (or H), an arginine/lysine carboxypeptidase with anacid pH optimum, is located in secretory granules of pancreatic islets,adrenal gland, pituitary, and brain. Zuhlke et al., Ciba Found. Symp.,41: 183-195 (1975); Davidson and Hutton, Biochem. J., 245: 575-582(1987); Hook and Loh, Proc. Natl. Acad. Sci. USA, 81: 2776-2780 (1984).It is believed that this enzyme removes the residual COOH-terminal Argor Lys remaining after initial endoprotease cleavage during prohormoneprocessing at the intragranular acid pH. Fricker, Annu. Rev. Physiol.,50: 309-321 (1988). Carboxypeptidase E has been isolated, cloned, andsequenced from different sources (rat: Frickler et al., J. Mol.Endocrinol., 3: 666-673 [1989]; bovine: Fricker et al., Nature, 323:461-464 [1986]; human: Hook and Affolter, FEBS Lett., 238: 338-342[1988]; Manser et al., Biochem, J., 267: 517-525 [1990]).

Carboxypeptidase M is a membrane-bound arginine/lysine carboxypeptidasefound in many tissues and cultured cells. Skidgel, Trends Pharmacol.Sci., 9: 299-304 (1988). Recently, it has been purified to homogeneityfrom human placenta. Skidgel et al., J. Biol. Chem., 264: 2236-2241(1989). Because of its presence on plasma membranes and optimal activityat neutral pH, it may act on peptide hormones at local tissue siteswhere it could control their activity before or after interaction withspecific plasma membrane receptors. Sequencing has showncarboxypeptidase M to be a unique enzyme that exhibits some similarityto carboxypeptidases A, B, E, and N. Tan et al., J. Biol. Chem., 264:13165-13170 (1989).

It is an object of the present invention to identify a novelcarboxypeptidase B isolated from plasma that shares some commonstructural features and catalytic and substrate binding sites withcarboxypeptidase A and with pancreas carboxypeptidase B, and in itshuman embodiment shares the most sequence identity with knowncarboxypeptidases A and B.

It is another object to provide nucleic acid encoding such a polypeptideand to use this nucleic acid to produce the polypeptide in recombinantcell culture for diagnostic use or for potential therapeutic use inhemostatic regulation.

It is yet another object to provide derivatives and modified forms ofsuch a new polypeptide, including amino acid sequence variants andcovalent derivatives thereof.

It is an additional object to prepare immunogens for raising antibodiesagainst such new polypeptide, as well as to obtain antibodies capable ofbinding it.

These and other objects of the invention will be apparent to theordinary artisan upon consideration of the specification as a whole.

SUMMARY OF THE INVENTION

These objects are accomplished, in one aspect, by providing an isolatednovel polypeptide that binds to plasminogen and is related structurallyand functionally to carboxypeptidase A and pancreas carboxypeptidase B.This polypeptide is hereafter termed plasma carboxypeptidase B (PCPB),and includes N-terminal and C-terminal fragments thereof.

In another aspect, the invention provides a composition comprising thePCPB that is free of contaminating polypeptides of the animal speciesfrom which the PCPB is derived.

PCPB or fragments thereof (which also may be synthesized by in vitromethods) are fused (by recombinant expression or in vitro covalentmethods) to an immunogenic polypeptide and this fusion polypeptide, inturn, is used to immunize an animal to raise antibodies against a PCPBepitope. Anti-PCPB is recovered from the serum of immunized animals.Alternatively, monoclonal antibodies are prepared from cells of theimmunized animal in conventional fashion. Antibodies identified byroutine screening will bind to PCPB but will not substantiallycross-react with any other known carboxypeptidase, includingcarboxypeptidase A, pancreas carboxypeptidase B, or carboxypeptidases E,M, and N, or carboxypeptidases from non-mammalian sources.

Immobilized anti-PCPB antibodies are useful particularly in thediagnosis (in vitro or in vivo) or purification of PCPB, e.g., a mixtureof PCPB is passed over a column to which the antibodies are bound.

Substitutional, deletional, or insertional variants of PCPB are preparedby in vitro or recombinant methods and screened forimmuno-crossreactivity with PCPB and for PCPB antagonist or agonistactivity.

PCPB also is derivatized in vitro to prepare immobilized PCPB andlabeled PCPB, particularly for purposes of diagnosis of PCPB or itsantibodies, or for affinity purification of PCPB antibodies.

PCPB, its derivatives, or its antibodies are formulated intophysiologically acceptable vehicles, especially for therapeutic use.Such vehicles include sustained-release formulations of PCPB.

In further aspects, the invention provides a method for purifying PCPBcomprising passing a mixture of PCPB over a plasminogen column,preferably eluted with epsilon-aminocaproic acid, or over a column towhich PCPB antibodies are bound and recovering the fraction containingPCPB. Also provided is a method for coagulating blood comprising addingto the blood an effective amount of PCPB, wherein in one embodiment themethod involves the in vivo treatment of a mammal (e.g., human) with ablood clotting disorder such as hemophilia.

In still other aspects, the invention provides an isolated nucleic acidmolecule encoding PCPB, labeled or unlabeled, and a nucleic acidsequence that is complementary, or hybridizes under stringent conditionsto, a nucleic acid sequence encoding PCPB, excluding nucleic acidsequences complementary to nucleic acid sequences encoding acarboxypeptidase A, a non-plasma, e.g., pancreas, carboxypeptidase B, acarboxypeptidase E, M, or N, or a non-mammalian carboxypeptidase, i.e.,those known carboxypeptidases that are not PCPB.

In addition, the invention provides a replicable vector comprising thenucleic acid molecule encoding PCPB operably linked to control sequencesrecognized by a host transformed by the vector; host cells transformedwith the vector; and a method of using a nucleic acid molecule encodingPCPB to effect the production of PCPB, comprising expressing the nucleicacid molecule in a culture of the transformed host cells and recoveringPCPB from the host cell culture. The nucleic acid sequence is alsouseful in hybridization assays for PCPB nucleic acid.

In still further embodiments, the invention provides a method forproducing PCPB comprising inserting into the DNA of a cell containingthe nucleic acid molecule encoding PCPB a transcription modulatoryelement in sufficient proximity and orientation to the nucleic acidmolecule to influence transcription thereof, with an optional furtherstep comprising culturing the cell containing the transcriptionmodulatory element and the nucleic acid molecule.

In still further embodiments, the invention provides a cell comprisingthe nucleic acid molecule encoding PCPB and an exogenous transcriptionmodulatory element in sufficient proximity and orientation to thenucleic acid molecule to influence transcription thereof; and a hostcell containing the nucleic acid molecule encoding PCPB operably linkedto exogenous control sequences recognized by the host cell.

Still further is provided a method for obtaining cells having increasedor decreased transcription of the nucleic acid molecule encoding PCPBcomprising:

(a) providing cells containing the nucleic acid molecule;

(b) introducing into the cells a transcription modulating element; and

(c) screening the cells for a cell in which the transcription of thenucleic acid molecule is increased or decreased.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a graph of optical density at 405 nm for mixtures ofplasminogen and t-PA with and without PCPB and with and withoutfibrinogen ("fibrin.").

FIG. 2 illustrates a graph of optical density at 405 nm for plasmin("CONT."), plasmin plus PCPB ("PCPB"), and plasmin plus anti-plasmin("AP").

FIG. 3 illustrates a restriction map of pUC218, which is prepared byinserting the indicated nucleotides into the designated site of pUC118.

FIGS. 4A-4D depict the nucleotide sequence for the human PCPB gene andthe deduced amino acid sequence. The arrow and positive numbers indicatewhere the mature sequence begins. A 46-mer sequence used to obtainfull-length clones is also shown, as well as the potential clip site(arginine) for activation of PCPB as a carboxypeptidase, indicated bytriple arrows. In addition, the expected residues involved in catalyticactivity are in bold, and the expected residues involved in substratebinding are indicated by double underlining, with the expected residuethat determines specificity of the PCPB as a carboxypeptidase B (Asp at348) also indicated by italics.

FIGS. 5A-5C align the amino acid sequences among human PCPB (hpcpb), ratcarboxypeptidase B (rcpb), rat carboxypeptidase A1 (rtcpa), human mastcell carboxypeptidase A (hmccpa), mouse mast cell carboxypeptidase A(mmccpa), and rat carboxypeptidase A2 (rtcpa2) to show the extent ofsequence identity. The potential substrate binding site for thecarboxypeptidases is indicated with an arrow.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In general, the following words or phrases have the indicated definitionwhen used in the description, examples, and claims:

The expression "control sequences" refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, aribosome binding site, and possibly, other as yet poorly understoodsequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

An "exogenous" element is defined herein to mean foreign to the cell, orhomologous to the cell but in a position within the host cell in whichthe element is ordinarily not found.

As used herein, the expressions "cell," "cell line," and "cell culture"are used interchangeably and all such designations include progeny.Thus, the words "transformants" and "transformed cells" include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same functionality as screenedfor in the originally transformed cell are included. Where distinctdesignations are intended, it will be clear from the context.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein arecommercially available, are publicly available on an unrestricted basis,or can be constructed from such available plasmids in accord withpublished procedures. In addition, other equivalent plasmids are knownin the art and will be apparent to the ordinary artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with anenzyme that acts only at certain locations in the DNA. Such enzymes arecalled restriction enzymes, and the site for which each is specific iscalled a restriction site. The various restriction enzymes used hereinare commercially available and their reaction conditions, cofactors, andother requirements as established by the enzyme suppliers are used.Restriction enzymes commonly are designated by abbreviations composed ofa capital letter followed by other letters representing themicroorganism from which each restriction enzyme originally was obtainedand then a number designating the particular enzyme. In general, about 1μg of plasmid or DNA fragment is used with about 1-2 units of enzyme inabout 20 μl of buffer solution. Appropriate buffers and substrateamounts for particular restriction enzymes are specified by themanufacturer. Incubation of about 1 hour at 37° C. is ordinarily used,but may vary in accordance with the supplier's instructions. Afterincubation, protein or polypeptide is removed by extraction with phenoland chloroform, and the digested nucleic acid is recovered from theaqueous fraction by precipitation with ethanol. Digestion with arestriction enzyme infrequently is followed with bacterial alkalinephosphatase hydrolysis of the terminal 5' phosphates to prevent the tworestriction cleaved ends of a DNA fragment from "circularizing" orforming a closed loop that would impede insertion of another DNAfragment at the restriction site. Unless otherwise stated, digestion ofplasmids is not followed by 5' terminal dephosphorylation. Proceduresand reagents for dephosphorylation are conventional (Maniatis et al.,Molecular Cloning: A Laboratory Manual [New York: Cold Spring HarborLaboratory, 1982] pp. 133-134).

"Recovery" or "isolation" of a given fragment of DNA from a restrictiondigest means separation of the digest on polyacrylamide or agarose gelby electrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. This procedure is knowngenerally. For example, see Lawn et al., Nucleic Acids Res., 9:6103-6114 (1981), and Goeddel et al., Nucleic Acids Res. 8: 4057 (1980).

"Southern analysis" is a method by which the presence of DNA sequencesin a digest or DNA-containing composition is confirmed by hybridizationto a known, labeled oligonucleotide or DNA fragment. For the purposesherein, unless otherwise provided, Southern analysis shall meanseparation of digests on 1 percent agarose, denaturation, and transferto nitrocellulose by the method of Southern, J. Mol. Biol., 98: 503-517(1975), and hybridization as described by Maniatis et al., Cell 15:687-701 (1978).

"Ligation" refers to the process of forming phospho-diester bondsbetween two double-stranded nucleic acid fragments (Maniatis et al.,1982, supra, p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units of T4 DNAligase ("ligase") per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

"Preparation" of DNA from transformants means isolating plasmid DNA frommicrobial culture. Unless otherwise provided, the alkaline/SDS method ofManiatis et al., 1982, supra, p. 90, may be used.

"Oligonucleotides" are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as described in EP 266,032 published 4 May1988, or via deoxynucleoside H-phosphonate intermediates as described byFroehler et al., Nucl. Acids Res., 14: 5399-5407 [1986]). They are thenpurified on polyacrylamide gels.

The technique of "polymerase chain reaction," or "PCR," as used hereingenerally refers to a procedure wherein minute amounts of a specificpiece of DNA are amplified as described in U.S. Pat. No. 4,683,195issued 28 Jul. 1987. Generally, sequence information from the ends ofthe stretch of interest or beyond needs to be available, such thatoligonucleotide primers can be designed; these primers will pointtowards one another, and will be identical or similar in sequence toopposite strands of the template to be amplified. The 5' terminalnucleotides of the two primers will coincide with the ends of theamplified material. PCR can be used to amplify specific DNA sequencesfrom total genomic DNA, cDNA transcribed from total cellular RNA,bacteriophage or plasmid sequences, etc. See generally Mullis et al.,Cold Spring Harbor Symp. Quant. Biol., 51: 263 (1987); Erlich, ed., PCRTechnology, (Stockton Press, NY, 1989).

"PCPB" is defined to be a polypeptide or protein encoded by the humanPCPB nucleotide sequence set forth in FIG. 4; a polypeptide that is thetranslated amino acid sequence set forth in FIG. 4; a polypeptide thatis the translated mature amino acid sequence shown in FIG. 4; fragmentsthereof having greater than about 5 residues comprising an immuneepitope or other biologically active site of PCPB (such as theN-terminal activation peptide fragment from +1 to +92 of the FIG. 4sequence numbered starting at the mature N-terminal phenylalanineresidue, and the C-terminal carboxypeptidase-active fragment from +93 to+401 of the FIG. 4 amino acid sequence); amino acid sequence variants ofsaid FIG. 4 sequence wherein an amino acid residue has been inserted N-or C-terminal to, or within, said FIG. 4 sequence or its fragment asdefined above; and/or amino acid sequence variants of said FIG. 4sequence or its fragment as defined above wherein an amino acid residueof said FIG. 4 sequence or fragment thereof has been substituted byanother residue, including predetermined mutations by, e.g.,site-directed or PCR mutagenesis, and other animal species of PCPB suchas rat, porcine, non-human primate, equine, murine, and ovinepreproPCPB, and alleles and other naturally occurring variants of theforegoing and human sequences; and derivatives of PCPB or its fragmentsas defined above wherein the PCPB or its fragments have been covalentlymodified by substitution with a moiety other than a naturally occurringamino acid. Such fragments and variants exclude any polypeptideheretofore identified, including any known carboxypeptidase of anyanimal species or any known fragment of such carboxypeptidase, includingplasma, mast cell, or pancreas carboxypeptidase A, pancreascarboxypeptidase B, and carboxypeptidases E, M, and N, and non-mammaliancarboxypeptidases such as plant, insect, fish, yeast, and bacterialcarboxypeptidases. PCPB amino acid sequence variants generally willshare at least about 75% (preferably >80%, more preferably >85%)sequence identity with the translated sequence shown in FIG. 4, afteraligning (introducing any necessary spaces) to provide maximum homologyand not considering any conservative substitutions as part of thesequence identity. Neither N- nor C-terminal extensions nor insertionsshall be construed as reducing homology.

The PCPB herein is capable of immunologically crossreacting with apolyclonal antibody raised to native PCPB and/or is positive in one ofthe following two bioassays: it has no effect on plasmin activity in anassay using the chromogenic plasmin substrate S-2251 assay as describedin the examples; and/or it blocks conversion of plasminogen to plasminin the presence of fibrinogen fragments and tissue plasminogen activatorusing the S-2251 assay where a 1:1 molar ratio of candidate polypeptideto plasminogen is employed.

The preferred PCPB is human PCPB, which more preferably has a molecularweight on non-reducing SDS-PAGE of about 60 kD and has in its matureform the N-terminal sequence PheGlnSer.

In one embodiment, PCPB is purified from human plasma (preferably anammonium sulfate precipitated fraction from plasma) or from transformedcell culture (lysed cells or supernatant) by passing a mixture of thePCPB over a column to which plasminogen is bound, such as a controlledglass pore column, and recovering the fraction containing the PCPB byelution and washing with appropriate solvent(s). Preferably the columnis eluted with epsilon-aminocaproic acid (EACA), and more preferably thecolumn is eluted with 0.2M EACA or a gradient of 0 to 50 mM EACA. If thePCPB is being purified from a native source, after the plasminogencolumn treatment, the PCPB fraction is preferably passed over a columnto which protein-A is bound to remove any contaminating immunoglobulins,and the fraction containing PCPB is recovered. The thus-recoveredfraction is then preferably passed over another column to whichplasminogen is bound using a 0 to 50 mM gradient of EACA, and thePCPB-containing fraction is recovered.

"Isolated" PCPB is PCPB that is identified and separated fromcontaminant polypeptides in the animal or human source of the PCPB.

Amino acid sequence variants of PCPB are prepared by introducingappropriate nucleotide changes into the PCPB DNA, or by in vitrosynthesis of the desired PCPB. Such variants include, for example,deletions from, or insertions or substitutions of, residues within theamino acid sequence shown for human PCPB in FIGS. 4A-4D. Any combinationof deletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. The amino acid changes also may result in furthermodifications of PCPB upon expression in recombinant hosts, e.g.introducing or moving sites of glycosylation, or introducing membraneanchor sequences (in accordance with PCT WO 89/01041 published 9 Feb.1989).

There are two principal variables in the construction of amino acidsequence variants: the location of the mutation site and the nature ofthe mutation. These are variants from the FIG. 4 sequence, and mayrepresent naturally occurring alleles (which will not requiremanipulation of the PCPB DNA) or predetermined mutant forms made bymutating the DNA, either to arrive at an allele or a variant not foundin nature. In general, the location and nature of the mutation chosenwill depend upon the PCPB characteristic to be modified.

For example, candidate PCPB antagonists (suitable as adjuncts tothrombolytic therapy such as t-PA) or super agonists will be initiallyselected by locating sites that are identical or highly conserved amongPCPB and known carboxypeptidases, especially among carboxypeptidases A,B and N. These sites then will be modified in series, e.g., by (1)substituting first with conservative choices and then with more radicalselections depending upon the results achieved, (2) deleting the targetresidue, or (3) inserting residues of the same or a different classadjacent to the located site, or combinations of options 1-3.

One helpful technique is called "alanine scanning mutagenesis." Here, aresidue or group of target residues are identified (e.g., chargedresidues such as arg, asp, his, lys, and glu) and replaced by a neutralor negatively charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids with thesurrounding aqueous environment in or outside the cell. Cunningham andWells, Science, 244: 1081-1085 (1989). Those domains demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at or for the sites ofsubstitution.

Obviously, such variations that, for example, convert PCPB into a knowncarboxypeptidase such as plasma, pancreas, or mast cell carboxypeptidaseA, pancreas carboxypeptidase B, plasma carboxypeptidase N,carboxypeptidases E or M, or non-mammalian carboxypeptidases are notincluded within the scope of this invention, nor are any other PCPBvariants or polypeptide sequences that are not novel and unobvious overthe prior art. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to optimize the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed PCPB variantsare screened for the optimal combination of desired activity.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Contiguous deletions ordinarily are made in even numbers ofresidues, but single or odd numbers of deletions are within the scopehereof. Deletions may be introduced into regions of low homology amongPCPB and carboxypeptidases A and B (which share the most sequenceidentity to the human PCPB amino acid sequence) to modify the activityof PCPB. Deletions from PCPB in areas of substantial homology withcarboxypeptidases A and B will be more likely to modify the biologicalactivity of PCPB more significantly. The number of consecutive deletionswill be selected so as to preserve the tertiary structure of PCPB in theaffected domain, e.g., beta-pleated sheet or alpha helix.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Intrasequence insertions (i.e.,insertions within the mature PCPB sequence) may range generally fromabout 1 to 10 residues, more preferably 1 to 5, most preferably 1 to 3.Insertions are preferably made in even numbers of residues, but this isnot required. Examples of terminal insertions include mature PCPB withan N-terminal methionyl residue, an artifact of the direct expression ofmature PCPB in recombinant cell culture, and fusion of a heterologousN-terminal signal sequence to the N-terminus of the mature PCPB moleculeto facilitate the secretion of mature PCPB from recombinant hosts. Suchsignals generally will be homologous to the intended host cell andinclude STII or lpp for E. coli, alpha factor for yeast, and viralsignals such as herpes gD for mammalian cells. Other insertions includethe fusion to the N- or C-terminus of PCPB of immunogenic polypeptides,e.g., bacterial polypeptides such as beta-lactamase or an enzyme encodedby the E. coli trp locus, or yeast protein, and C-terminal fusions withproteins having a long half-life such as immunoglobulin constantregions, albumin, or ferritin, as described in WO 89/02922 published 6Apr. 1989.

The third group of variants are those in which at least one amino acidresidue in the PCPB molecule, and preferably only one, has been removedand a different residue inserted in its place. The sites of greatestinterest for substitutional mutagenesis include sites where the aminoacids found in known carboxypeptidases A and/or B and novel PCPB aresubstantially different in terms of side-chain bulk, charge, orhydrophobicity, but where there also is a high degree of sequenceidentity at the selected site within various animal analogues ofcarboxypeptidase A or carboxypeptidase B (e.g., among all the animalcarboxypeptidase A molecules or among all the animal carboxypeptidase Bmolecules). This analysis will highlight residues that may be involvedin the differentiation of activity of the carboxypeptidases, andtherefore, variants at these sites may affect such activities.

Other sites of interest are those in which the residues are identicalamong all animal species of PCPB and carboxypeptidase A or non-plasmaderived carboxypeptidase B, this degree of conformation suggestingimportance in achieving biological activity common to these enzymes.These sites, especially those falling within a sequence of at leastthree other identically conserved sites, are substituted in a relativelyconservative manner. Such conservative substitutions are shown in Table1 under the heading of preferred substitutions. If such substitutionsresult in a change in biological activity, then more substantialchanges, denominated exemplary substitutions in Table 1, or as furtherdescribed below in reference to amino acid classes, are introduced andthe products screened.

                  TABLE 1                                                         ______________________________________                                        Original   Exemplary        Preferred                                         Residue    Substitutions    Substitutions                                     ______________________________________                                        Ala (A)    val; leu; ile    val                                               Arg (R)    lys; gln; asn    lys                                               Asn (N)    gln; his; lys; arg                                                                             gln                                               Asp (D)    glu              glu                                               Cys (C)    ser              ser                                               Gln (Q)    asn              asn                                               Glu (E)    asp              asp                                               Gly (G)    pro              pro                                               His (H)    asn; gln; lys; arg                                                                             arg                                               Ile (I)    leu; val; met; ala; phe;                                                      norleucine       leu                                               Leu (L)    norleucine; ile; val;                                                         met; ala; phe    ile                                               Lys (K)    arg; gln; asn    arg                                               Met (M)    leu; phe; ile    leu                                               Phe (P)    leu; val; ile; ala                                                                             leu                                               Pro (P)    gly              gly                                               Ser (S)    thr              thr                                               Thr (T)    ser              ser                                               Trp (W)    tyr              tyr                                               Tyr (Y)    trp; phe; thr; ser                                                                             phe                                               Val (V)    ile; leu; met; phe;                                                                            leu                                                          ala; norleucine                                                    ______________________________________                                    

Trypsin or other protease cleavage sites are identified by inspection ofthe encoded amino acid sequence for an arginyl or lysinyl residue. Theseare rendered inactive to protease by substituting the residue withanother residue, preferably a basic residue such as glutamine or ahydrophobic residue such as serine; by deleting the residue; or byinserting a prolyl residue immediately after the residue.

In another embodiment, any methionyl residues other than the startingmethionyl residue of the signal sequence, or any residue located withinabout three residues N- or C-terminal to each such methionyl residue, issubstututed by another residue (preferably in accord with Table 1) ordeleted. Alternatively, about 1-3 residues are inserted adjacent to suchsites.

Sites particularly suited for conservative substitutions include,numbered from the N-terminus of the mature PCPB, P135, L136, Y137, V138,L139, K140, A151, I152, W153, I154, D155, G157, I158, A160, W163, I164,S165, P166, A167, F168, N202, V203, D204, G205, Y206, Y208, S209, W210,K211, K212, N213, R214, M215, W216, R217, K218, N219, R220, I229, G230,T231, D232, L233, N234, R235, N236, F237, P261, E262, E264, E266, V267,K268, A269, V270, I281, K282, A283, Y284, I285, H288, Y290, Q292, Y352,D353, L354, G355, I356, K357, Y358, F360, T361, E363, L364, R365, D366,T367, G368, G371, L373, L374, P375, E376, I379, K380, P381, T382, R384,and E385.

It is noted that the histidine residues at positions 159 and 288, theglutamic acid at position 162, and the glycine at position 347, all ofwhich except the glycine are conserved among the carboxypeptidasesdepicted in FIG. 5, are catalytic sites that are preferably not altered,e.g., by substitution or deletion. Also, the arginine residue atposition 161, the asparagine residue at position 234, the arginineresidue at position 235, the tyrosine residue at position 290, theaspartic acid residues at positions 348 and 349, and the phenylalanineresidue at position 372 are substrate binding sites that are notgenerally altered as by substitution with another amino acid or bydeletion, particularly the aspartic acid at position 348, which isbelieved to determine substrate specificity of PCPB as acarboxypeptidase B (see FIG. 5).

Any cysteine residues not involved in maintaining the properconformation of PCPB also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Sites other than those set forth in this paragraph aresuitable for deletional or insertional studies generally describedabove.

Substantial modifications in function or immunological identity areaccomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another. Such substituted residues also may beintroduced into the conservative substitution sites set forth above or,more preferably, into the remaining (non-conserved) sites.

Examples of PCPB variants include PCPB(150NAI152→NAS or NAT) (this addsan N-linked glycosylation site); PCPB(R11-Q83); PCPB(G4-C69) (variantsso depicted are fragments containing the residues indicated);PCPB(G4-K59); PCPB(C69-C156); PCPB(C69-C169); PCPB(C69-C228);PCPB(C69-C243); PCPB(C69-C252); PCPB(R12-R92); PCPB(R12-R117);PCPB(R92-R117); PCPB(C156-C169); PCPB(C169-C228); PCPB(C169C-243);PCPB(C169-C252); PCPB(C169-C257); PCPB(C169-C383); PCPB(C69-C257);PCPB(C69-C383); PCPB(R12-C156); PCPB(R92R-384); PCPB(R92-R275);PCPB(R92-R330); PCPB(K44-K124); PCPB(R12-K124); PCPB(R12-K44);PCPB(K124-K282); PCPB(G4-C69) E I L I H D V E D L; PCPB(G4-Q83) F D V KE; PCPB(R11-Q83) F D S H T; PCPB(R92-R399) H T S; PCPB(R92-R399) H L Y;PCPB(R92-R384) E T M L A V K; PCPB(R92-K392) I A K Y I L K H T S;PCPB(R92-K392) I A N Y V R E H L Y; PCPB(ΔC69); PCPB(ΔC156);PCPB(ΔC169-ΔC383) (variants depicted in this fashion comprise deletionsof the indicated span of residues, inclusive); W153→F; I154→M; I158→F;I164→V; L170→Q; V201→F or T; D207→I or V; Y208→W; K211→T; N225→G; I229→Lor V; T231→V; L233→P; P265→K or V; V267→T; S272→D; L274→I; L354→Q;Y358→H; S359→T; T369→K or F; Y370→F or R; R377→S; and Y378→R or Q.

Covalent modifications of PCPB molecules are included within the scopeof this invention. Variant PCPB fragments having up to about 40 residuesmay be conveniently prepared by in vitro synthesis. In addition,covalent modifications are introduced into the molecule by reactingtargeted amino acid residues of the PCPB with an organic derivatizingagent that is capable of reacting with selected side chains or the N- orC-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Parabromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablemethods for derivatizing α-amino-containing residues include use ofimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; and 2,4-pentanedione; and transaminase-catalyzedreaction with glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵ I or ¹³¹ I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R'--N═C═N--R'), where R and R' aredifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking PCPBto a water-insoluble support matrix or surface for use in the method forpurifying anti-PCPB antibodies, and vice versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group. PCPB also is covalently linked to nonproteinaceouspolymers, e.g. polyethylene glycol, polypropylene glycol orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

PCPB "nucleic acid" is defined as RNA or DNA that encodes a PCPB, iscomplementary to nucleic acid sequence encoding PCPB, hybridizes to suchnucleic acid and remains stably bound to it under stringent conditions,or encodes a polypeptide sharing at least 75% sequence identity,preferably at least 80%, and more preferably at least 85%, with thetranslated amino acid sequence shown in FIG. 4. It is typically at leastabout 10 bases in length and preferably has PCPB biological orimmunological activity, including the nucleic acid encoding anactivation peptide fragment having the nucleotide sequence shown in FIG.4 beginning at the codon for phenylalanine at the mature N-terminus andending at the codon for arginine at position 92, and the nucleic acidencoding a carboxypeptidase-active fragment having the nucleotidesequence shown in FIG. 4 beginning at the codon for alanine at position93 and ending at the codon for the valine at position 401. Suchhybridizing or complementary nucleic acid, however, is defined furtheras being novel and unobvious over any prior art nucleic acid includingthat which encodes, hybridizes under stringent conditions, or iscomplementary to nucleic acid encoding a known carboxypeptidase,including plasma, pancreas, and mast cell carboxypeptidase A, non-plasmaderived carboxypeptidase B, carboxypeptidases E, M, or N, and anon-mammalian carboxypeptidase.

"Stringent conditions" are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0.015M NaCl/0.0015M sodiumcitrate/0.1% NaDodSO₄ at 50° C., or (2) employ during hybridization adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C. Another example is use of 50% formamide, 5×SSC (0.75MNaCl, 0.075M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA(50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at42° C. in 0.2×SSC and 0.1% SDS.

DNA encoding PCPB is obtained from a liver cDNA library, or genomic DNA,or by in vitro synthesis. Hybridizing nucleic acid generally is obtainedby in vitro synthesis. Identification of PCPB DNA most conveniently isaccomplished by probing human cDNA or genomic libraries by labeledoligonucleotide sequences selected from the FIG. 4 sequence in accordwith known criteria, among which is that the sequence should be ofsufficient length and sufficiently unambiguous that false positives areminimized. Typically, a ³² P-labeled oligonucleotide having about 30 to50 bases is sufficient, particularly if the oligonucleotide contains oneor more codons for methionine or tryptophan. "Isolated" nucleic acidwill be nucleic acid that is identified and separated from contaminantnucleic acid encoding other polypeptides from the source of nucleicacid. The nucleic acid may be labeled for diagnostic and probe purposes,using a label as described and defined further below in the discussionof diagnostic assays.

Of particular interest is PCPB nucleic acid that encodes a full-lengthmolecule, including but not necessarily the native signal sequencethereof. Nucleic acid encoding full-length protein is obtained byscreening selected cDNA (not kidney) or genomic libraries using thededuced amino acid sequence disclosed herein for the first time, and, ifnecessary, using conventional primer extension procedures to secure DNAthat is complete at its 5' coding end. Such a clone is readilyidentified by the presence of a start codon in reading frame with theoriginal sequence.

DNA encoding amino acid sequence variants of PCPB is prepared by avariety of methods known in the art. These methods include, but are notlimited to, isolation from a natural source (in the case of naturallyoccuring amino acid sequence variants) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of PCPB.

Oligonucleotide-mediated mutagenesis is a preferred method for preparingsubstitution, deletion, and insertion variants of PCPB DNA. Thistechnique is well known in the art as described by Adelman et al., DNA,2: 183 (1983). Briefly, PCPB DNA is altered by hybridizing anoligonucleotide encoding the desired mutation to a DNA template, wherethe template is the single-stranded form of the plasmid containing theunaltered or native DNA sequence of PCPB. After hybridization, a DNApolymerase is used to synthesize an entire second complementary strandof the template that will thus incorporate the oligonucleotide primer,and will code for the selected alteration in the PCPB DNA.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., Proc.Natl. Acad. Sci. USA, 75: 5765 (1978).

The DNA template can only be generated by those vectors that are eitherderived from bacteriophage M13 vectors (the commercially availableM13mp18 and M13mp19 vectors are suitable), or those vectors that containa single-stranded phage origin of replication as described by Viera etal. Meth. Enzymol., 153: 3 (1987). Thus, the DNA that is to be mutatedmust be inserted into one of these vectors to generate single-strandedtemplate. Production of the single-stranded template is described inSections 4.21-4.41 of Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory Press, NY 1989).

For alteration of the native DNA sequence, the oligonucleotide ishybridized to the single-stranded template under suitable hybridizationconditions. A DNA polymerizing enzyme, usually the Klenow fragment ofDNA polymerase I, is then added to synthesize the complementary strandof the template using the oligonucleotide as a primer for synthesis. Aheteroduplex molecule is thus formed such that one strand of DNA encodesthe mutated form of PCPB, and the other strand (the original template)encodes the native, unaltered sequence of PCPB. This heteroduplexmolecule is then transformed into a suitable host cell, usually aprokaryote such as E. coli JM101. After the cells are grown, they areplated onto agarose plates and screened using the oligonucleotide primerradiolabeled with 32-phosphate to identify the bacterial colonies thatcontain the mutated DNA. The mutated region is then removed and placedin an appropriate vector for protein production, generally an expressionvector of the type typically employed for transformation of anappropriate host.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: Thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTTP), is combined with a modifiedthio-deoxyribocytosine called dCTP-(aS) (which can be obtained fromAmersham). This mixture is added to the template-oligonucleotidecomplex. Upon addition of DNA polymerase to this mixture, a strand ofDNA identical to the template except for the mutated bases is generated.In addition, this new strand of DNA will contain dCTP-(aS) instead ofdCTP, which serves to protect it from restriction endonucleasedigestion.

After the template strand of the double-stranded heteroduplex is nickedwith an appropriate restriction enzyme, the template strand can bedigested with ExoIII nuclease or another appropriate nuclease past theregion that contains the site(s) to be mutagenized. The reaction is thenstopped to leave a molecule that is only partially single-stranded. Acomplete double-stranded DNA homoduplex is then formed using DNApolymerase in the presence of all four deoxyribonucleotidetriphosphates, ATP, and DNA ligase. This homoduplex molecule can then betransformed into a suitable host cell such as E. coli JM101, asdescribed above.

Mutants with more than one amino acid to be substituted may be generatedin one of several ways. If the amino acids are located close together inthe polypeptide chain, they may be mutated simultaneously using oneoligonucleotide that codes for all of the desired amino acidsubstitutions. If, however, the amino acids are located some distancefrom each other (separated by more than about ten amino acids), it ismore difficult to generate a single oligonucleotide that encodes all ofthe desired changes. Instead, one of two alternative methods may beemployed.

In the first method, a separate oligonucleotide is generated for eachamino acid to be substituted. The oligonucleotides are then annealed tothe single-stranded template DNA simultaneously, and the second strandof DNA that is synthesized from the template will encode all of thedesired amino acid substitutions.

The alternative method involves two or more rounds of mutagenesis toproduce the desired mutant. The first round is as described for thesingle mutants: wild-type DNA is used for the template, anoligonucleotide encoding the first desired amino acid substitution(s) isannealed to this template, and the heteroduplex DNA molecule is thengenerated. The second round of mutagenesis utilizes the mutated DNAproduced in the first round of mutagenesis as the template. Thus, thistemplate already contains one or more mutations. The oligonucleotideencoding the additional desired amino acid substitution(s) is thenannealed to this template, and the resulting strand of DNA now encodesmutations from both the first and second rounds of mutagenesis. Thisresultant DNA can be used as a template in a third round of mutagenesis,and so on.

PCR mutagenesis is also suitable for making amino acid variants of PCPB.This technique refers to the following procedure (see Erlich, supra, thechapter by R. Higuchi, p. 61-70): When small amounts of template DNA areused as starting material in a PCR, primers that differ slightly insequence from the corresponding region in a template DNA can be used togenerate relatively large quantities of a specific DNA fragment thatdiffers from the template sequence only at the positions where theprimers differ from the template. For introduction of a mutation into aplasmid DNA, one of the primers is designed to overlap the position ofthe mutation and to contain the mutation; the sequence of the otherprimer must be identical to a stretch of sequence of the opposite strandof the plasmid, but this sequence can be located anywhere along theplasmid DNA. It is preferred, however, that the sequence of the secondprimer is located within 200 nucleotides from that of the first, suchthat in the end the entire amplified region of DNA bounded by theprimers can be easily sequenced. PCR amplification using a primer pairlike the one just described results in a population of DNA fragmentsthat differ at the position of the mutation specified by the primer, andpossibly at other positions, as template copying is somewhaterror-prone.

If the ratio of template to product material is extremely low, the vastmajority of product DNA fragments incorporate the desired mutation(s).This product material is used to replace the corresponding region in theplasmid that served as PCR template using standard DNA technology.Mutations at separate positions can be introduced simultaneously byeither using a mutant second primer, or performing a second PCR withdifferent mutant primers and ligating the two resulting PCR fragmentssimultaneously to the vector fragment in a three (or more)-partligation.

In a specific example of PCR mutagenesis, template plasmid DNA (1 μg) islinearized by digestion with a restriction endonuclease that has aunique recognition site in the plasmid DNA outside of the region to beamplified. Of this material, 1-5 ng is added to a PCR mixture containing16.6 mM (NH₄)₂ SO₄, 67 mM Tris.HCl (pH 8.8), 6.7 mM MgCl₂, 6.7 μM EDTA,10 mM 2-mercaptoethanol, 1 mM each dATP, dCTP, dGTP, and TTP, 170 μg/mlbovine serum albumin, 25 pmole of each oligonucleotide primer, and 1 μlThermus aquacicus (Taq) DNA polymerase (5 units/μl, purchased fromPerkin-Elmer Cetus, Norwalk, Conn. and Emeryville, Calif.) in a finalvolume of 50 μl in a 0.5-ml reaction vial. The reaction mixture isoverlayed with 35 μl mineral oil and inserted into a DNA Thermal Cycler(purchased from Perkin-Elmer Cetus) programmed as follows:

    ______________________________________                                        time-delay file     12 min. 94° C.                                     thermo-cycle file   1 min. 50° C.                                                          2-3 min. 68-72° C.                                                     1 min. 94° C.                                                          20 cycles                                                 time-delay file     4 min. 50° C.                                      time-delay file     12 min. 68° C.                                     soak file           4° C.                                              ______________________________________                                    

Each file shown above is linked to the one on the next line. At the endof the program, the reaction vial is removed from the thermal cycler andthe aqueous phase transferred to a new vial, extracted withphenol/chloroform/isoamylalcohol (50:50:1 vol), and ethanolprecipitated, and the DNA is recovered by standard procedures. Thismaterial is subsequently subjected to the appropriate treatments forinsertion into a vector.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al., Gene, 34: 315 (1985). Thestarting material is the plasmid (or other vector) comprising the PCPBDNA to be mutated. The codon(s) in the PCPB DNA to be mutated areidentified. There must be a unique restriction endonuclease site on eachside of the identified mutation site(s). If no such restriction sitesexist, they may be generated using the above-describedoligonucleotide-mediated mutagenesis method to introduce them atappropriate locations in the PCPB DNA. After the restriction sites havebeen introduced into the plasmid, the plasmid is cut at these sites tolinearize it. A double-stranded oligonucleotide encoding the sequence ofthe DNA between the restriction sites but containing the desiredmutation(s) is synthesized using standard procedures. The two strandsare synthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 3' and 5' ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutatedPCPB DNA sequence.

The PCPB-encoding nucleic acid, whether variant or cDNA or genomic DNA,is ligated into a replicable vector for further cloning or forexpression. Vectors are useful for performing two functions incollaboration with compatible host cells (a host-vector system). Onefunction is to facilitate the cloning of the nucleic acid that encodesthe PCPB, i.e., to produce usable quantities of the nucleic acid. Theother function is to direct the expression of PCPB. One or both of thesefunctions are performed by the vector-host system. The vectors willcontain different components depending upon the function they are toperform as well as the host cell that is selected for cloning orexpression.

Particularly useful in the invention are expression vectors that providefor the transient expression in mammalian cells of DNA encoding PCPB. Ingeneral, transient expression involves the use of an expression vectorthat is able to replicate efficiently in a host cell, such that the hostcell accumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector. Sambrook et al., supra, pp. 16.17-16.22. Transientexpression systems, comprising a suitable expression vector and a hostcell, allow for the convenient positive identification of polypeptidesencoded by cloned DNAs, as well as for the rapid screening of suchpolypeptides for desired biological or physiological properties. Thus,transient expression systems are particularly useful in the inventionfor purposes of identifying analogs and variants of PCPB that havecarboxypeptidase or other PCPB-like activity.

Each expression vector will contain nucleic acid that encodes PCPB asdescribed above. The PCPBs of this invention are expressed directly inrecombinant cell culture as an N-terminal methionyl analogue, or as afusion with a heterologous polypeptide, preferably a signal sequence orother polypeptide having a specific cleavage site at the N-terminus ofthe mature protein or polypeptide. For example, in constructing aprokaryotic secretory expression vector for PCPB, the native PCPB signalis employed with hosts that recognize that signal. When the secretoryleader is "recognized" by the host, the host signal peptidase is capableof cleaving a fusion of the leader polypeptide fused at its C-terminusto the desired mature PCPB.

For host prokaryotes that do not process the PCPB signal, the signal issubstituted by a prokaryotic signal selected, for example, from thegroup of the alkaline phosphatase, penicillinase, lpp, or heat-stableenterotoxin II leaders. For yeast secretion the native signal may besubstituted by the yeast invertase, alpha factor, or acid phosphataseleaders. In mammalian cell expression the native signal (i.e., the PCPBpresequence that normally directs secretion of PCPB from human cells invivo) is satisfactory, although other mammalian secretory proteinsignals are suitable such as signals from other animal PCPBs, signalsfrom a carboxypeptidase A, E, M, or N or non-plasma derivedcarboxypeptidase B, and signals from secreted polypeptides of the sameor related species, as are viral secretory leaders, for example, theherpes simplex gD signal.

If the signal sequence is from another carboxypeptidase molecule, it maybe the precursor sequence spanning from the initiating methionine (M)residue shown in FIG. 5 of a carboxypeptidase A or B up to the alanine(A) residue just before the first amino acid of the mature protein, or aconsensus or combination sequence from any two or more of the precursorsof different carboxypeptidases taking into account homologous regions ofthe precursors. The DNA for such precursor region is ligated in readingframe to DNA encoding the mature PCPB.

Expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomes, and includesorigins of replication or autonomously replicating sequences. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells. Origins are not needed formammalian expression vectors (the SV40 origin may typically be used onlybecause it contains the early promoter). Most expression vectors are"shuttle" vectors, i.e. they are capable of replication in at least oneclass of organisms but can be transfected into another organism forexpression. For example, a vector is cloned in E. coli and then the samevector is transfected into yeast or mammalian cells for expression eventhough it is not capable of replicating independently of the host cellchromosome.

DNA also is cloned by insertion into the host genome. This is readilyaccomplished with Bacillus species, for example, by including in thevector a DNA sequence that is complementary to a sequence found inBacillus genomic DNA. Transfection of Bacillus with this vector resultsin homologous recombination with the genome and insertion of PCPB DNA.However, the recovery of genomic DNA encoding PCPB is more complex thanthat of an exogenously replicated vector because restriction enzymedigestion is required to excise the PCPB DNA.

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This is a gene that encodes a proteinnecessary for the survival or growth of a host cell transformed with thevector. The presence of this gene ensures that any host cell whichdeletes the vector will not obtain an advantage in growth orreproduction over transformed hosts. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins, e.g.ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g. the gene encoding D-alanine racemase forBacilli.

A suitable selection gene for use in yeast is the trpl gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 [1979];Kingsman et al., Gene, 7: 141 [1979]; or Tschemper et al., Gene, 10: 157[1980]). The trpl gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 (Jones, Genetics, 85: 12 [1977]). The presence ofthe trpl lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene express a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin (Southern et al., J. Molec. Appl. Genet., 1: 327[1982]), mycophenolic acid (Mulligan et al., Science, 209: 1422 [1980])or hygromycin (Sugden et al., Mol. Cell. Biol., 5: 410-413 [1985]). Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up thePCPB nucleic acid, such as dihydrofolate reductase (DHFR) or thymidinekinase. The mammalian cell transformants are placed under selectionpressure which only the transformants are uniquely adapted to survive byvirtue of having taken up the marker. Selection pressure is imposed byculturing the transformants under conditions in which the concentrationof selection agent in the medium is successively changed, therebyleading to amplification of both the selection gene and the DNA thatencodes PCPB. Amplification is the process by which genes in greaterdemand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Increased quantities of PCPB are synthesized from theamplified DNA.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216 [1980]. The transformed cells are then exposed toincreased levels of methotrexate. This leads to the synthesis ofmultiple copies of the DHFR gene, and, concomitantly, multiple copies ofother DNA comprising the expression vectors, such as the DNA encodingPCPB. This amplification technique can be used with any otherwisesuitable host, e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presenceof endogenous DHFR if, for example, a mutant DHFR gene that is highlyresistant to Mtx is employed (EP 117,060). Alternatively, host cells[particularly wild-type hosts that contain endogenous DHFR] transformedor co-transformed with DNA sequences encoding PCPB, wild-type DHFRprotein, and another selectable marker such as aminoglycoside 3'phosphotransferase (APH) can be selected by cell growth in mediumcontaining a selection agent for the selectable marker such as anaminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S.Pat. No. 4,965,199.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of PCPB in recombinant vertebrate cell culture are describedin Gething et al., Nature, 293: 620-625 [1981]; Mantei et al., Nature,281: 40-46 [1979]; Levinson et al.; EP 117,060; and EP 117,058. Aparticularly useful plasmid for mammalian cell culture expression ofPCPB is pRK5 (EP pub. no. 307,247) or pSVI6B (U.S. Ser. No. 07/441,574filed 22 Nov. 1989, the disclosure of which is incorporated herein byreference).

Expression vectors, unlike cloning vectors, should contain a promoterthat is recognized by the host organism and is operably linked to thePCPB nucleic acid. Promoters are untranslated sequences located upstreamfrom the start codon of a structural gene (generally within about 100 to1000 bp) that control the transcription and translation of nucleic acidunder their control. They typically fall into two classes, inducible andconstitutive. Inducible promoters are promoters that initiate increasedlevels of transcription from DNA under their control in response to somechange in culture conditions, e.g. the presence or absence of a nutrientor a change in temperature. At this time a large number of promotersrecognized by a variety of potential host cells are well known. Thesepromoters are operably linked to PCPB-encoding DNA by removing them fromtheir gene of origin by restriction enzyme digestion, followed byinsertion 5' to the start codon for PCPB. This is not to say that thegenomic PCPB promoter is not usable. However, heterologous promotersgenerally will result in greater transcription and higher yields ofexpressed PCPB.

Nucleic acid is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, "operably linked"means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. Linking isaccomplished by ligation at convenient restriction sites. If such sitesdo not exist, then synthetic oligonucleotide adaptors or linkers areused in accord with conventional practice.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature, 275: 615[1978]; and Goeddel et al., Nature, 281: 544 [1979]), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8: 4057 [1980] and EP 36,776) and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 [1983]).However, other known bacterial promoters are suitable. Their nucleotidesequences have been published, thereby enabling a skilled workeroperably to ligate them to DNA encoding PCPB (Siebenlist et al., Cell,20: 269 [1980]) using linkers or adaptors to supply any requiredrestriction sites. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding PCPB.

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem., 255: 2073 [1980]) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg., 7: 149 [1968]; and Holland, Biochemists, 17: 4900[1978]), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin Hitzeman et al., EP 73,657A. Yeast enhancers also are advantageouslyused with yeast promoters.

Expression control sequences are known for eukaryotes. Virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30bases upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3' end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3' end of the codingsequence. All of these sequences are suitably inserted into mammalianexpression vectors.

PCPB transcription from vectors in mammalian host cells is controlled bypromoters obtained from the genomes of viruses such as polyoma virus,fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g. theactin promoter or an immunoglobulin promoter, from heat-shock promoters,and from the promoter normally associated with the PCPB sequence,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 vital originof replication. Fiers et al., Nature, 273:113 (1978); Mulligan and Berg,Science, 209: 1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci.USA, 78: 7398-7402 (1981). The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. Greenaway et al., Gene, 18: 355-360 (1982). A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978. See also Gray et al.,Nature, 295: 503-508 (1982) on expressing cDNA encoding immuneinterferon in monkey cells, Reyes et al., Nature, 297: 598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus, Canaani andBerg, Proc. Natl. Acad. Sci. USA, 79: 5166-5170 (1982) on expression ofthe human interferon β1 gene in cultured mouse and rabbit cells, andGorman et al., Proc. Natl. Acad. Sci. USA, 79: 6777-6781 (1982) onexpression of bacterial CAT sequences in CV-1 monkey kidney cells,chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, andmouse NIH-3T3 cells using the Rous sarcoma virus long terminal repeat asa promoter.

Transcription of a DNA encoding the PCPB of this invention by highereukaryotes is increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription.Enhancers are relatively orientation and position independent havingbeen found 5' (Laimins et al., Proc. Natl. Acad. Sci. USA, 78: 993[1981]) and 3' (Lusky et al., Mol. Cell Bio. 3: 1108 [1983]) to thetranscription unit, within an intron (Banerji et al., Cell, 33: 729[1983]) as well as within the coding sequence itself (Osborne et al.,Mol. Cell Bio., 4: 1293 [1984]). Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature, 297: 17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5' or 3' to thePCPB-encoding sequence, but is preferably located at a site 5' from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5' and, occasionally 3' untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PCPB. The 3' untranslated regions alsoinclude transcription termination sites.

Suitable host cells for cloning or expressing the vectors herein are theprokaryote, yeast, or higher eukaryote cells described above. Suitableprokaryotes include eubacteria, such as Gram-negative or Gram-positiveorganisms, for example, E. coli, Bacilli such as B. subtilis,Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, orSerratia marcescans. One preferred E. coli cloning host is E. coli 294(ATCC 31,446), although other strains such as E. coli B, E. coli X1776(ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. Theseexamples are illustrative rather than limiting. Preferably the host cellshould secrete minimal amounts of proteolytic enzymes. Alternatively, invitro methods of cloning, e.g. PCR, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for PCPB-encoding vectors.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among lower eukaryotic host microorganisms. However, a number ofother genera, species, and strains are commonly available and usefulherein, such as S. pombe [Beach and Nurse, Nature, 290: 140 (1981)],Kluyveromyces lactis [Louvencourt et al., J. Bacteriol., 737 (1983)],yarrowia [EP 402,226], Pichia pastoris [EP 183,070], Trichoderma reesia[EP 244,234], Neurospora crassa [Case et al., Proc. Natl. Acad. Sci.USA, 76: 5259-5263 (1979)], and Aspergillus hosts such as A. nidulans[Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289 (1983);Tilburn et al., Gene, 26: 205-221 (1983); Yelton et al., Proc. Natl.Acad. Sci. USA, 81: 1470-1474 (1984)] and A. niger [Kelly and Hynes,EMBO J., 4: 475-479 (1985)].

Suitable host cells for the expression of PCPB are derived frommulticellular organisms. Such host cells are capable of complexprocessing and glycosylation activities. In principle, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosphila melanogaster (fruitfly), and Bombyx mori hostcells have been identified. See, e.g., Luckow et al., Bio/Technology, 6:47-55 (1988); Miller et al., in Genetic Engineering, Setlow, J. K. etal., eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda etal., Nature, 315: 592-594 (1985). A variety of such viral strains arepublicly available, e.g., the L-1 variant of Autographa californica NPVand the Bm-5 strain of Bombyx mori NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the PCPB DNA. During incubation of the plant cell culture withA. tumefaciens, the DNA encoding the PCPB is transferred to the plantcell host such that it is transfected, and will, under appropriateconditions, express the PCPB DNA. In addition, regulatory and signalsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.Depicker et al., J. Mol. Appl. Gen., 1: 561 (1982). In addition, DNAsegments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. EP321,196 published 21 Jun. 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure in recent years [Tissue culture, Academic Press, Kruse andPatterson, editors (1973)]. Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36: 59 [1977]); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 [1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 [1980]); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383: 44-68 [1982]); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction, and/or sequenced bythe method of Messing et al., Nucleic Acids Res., 9: 309 (1981) or bythe method of Maxam et al., Methods in Enzymology, 65: 499 (1980).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described by Cohen, Proc. Natl. Acad.Sci. (USA), 69: 2110 (1972) and Mandel et al., J. Mol. Biol., 53: 154(1970), is generally used for prokaryotes or other cells that containsubstantial cell-wall barriers. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23: 315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52: 456-457(1978) is preferred. General aspects of mammalian cell host systemtransformations have been described by Axel in U.S. Pat. No. 4,399,216issued 16 Aug. 1983. Transformations into yeast are typically carriedout according to the method of Van Solingen et al., J. Bact., 130: 946(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979).However, other methods for introducing DNA into cells such as by nuclearinjection or by protoplast fusion may also be used.

The mammalian host cells used to produce the PCPB of this invention maybe cultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham and Wallace, Meth. Enz., 58: 44 (1979), Barnes andSato, Anal. Biochem., 102: 255 (1980), U.S. Pat. Nos. 4,767,704;4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195; U.S. Pat.No. Re. 30,985; or copending U.S. Ser. Nos. 07/592,107 or 07/592,141,both filed on 3 Oct. 1990, the disclosures of all of which areincorporated herein by reference, may be used as culture media for thehost cells. Any of these media may be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleosides (such asadenosine and thymidine), antibiotics (such as Gentamycin™ drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

It is further envisioned that the PCPB of this invention may be producedby homologous recombination, or with recombinant production methodsutilizing control elements introduced into cells already containing DNAencoding the PCPB currently in use in the field. For example, a powerfulpromoter/enhancer element, a suppressor, or an exogenous transcriptionmodulatory element is inserted in the genome of the intended host cellin proximity and orientation sufficient to influence the transcriptionof DNA encoding the desired PCPB. The control element does not encodethe PCPB of this invention, but the DNA is present in the host cellgenome. One next screens for cells making the PCPB of this invention, orfor increased or decreased levels of expression, as desired.

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77: 5201-5205 [1980]), dot blotting (DNA analysis), orin situ hybridization, using an appropriately labeled probe, based onthe sequences provided herein. Various labels may be employed, mostcommonly radioisotopes, particularly ³² P. However, other techniques mayalso be employed, such as using biotin-modified nucleotides forintroduction into a polynucleotide. The biotin then serves as the sitefor binding to avidin or antibodies, which may be labeled with a widevariety of labels, such as radionuclides, fluorescers, enzymes, or thelike. Alternatively, antibodies may be employed that can recognizespecific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNAhybrid duplexes or DNA-protein duplexes. The antibodies in turn may belabeled and the assay may be carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hsu et al., Am. J. Clin. Path.,75: 734-738 (1980).

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal. Conveniently, theantibodies may be prepared against a synthetic peptide based on the DNAsequences provided herein as described further below.

PCPB preferably is recovered from the culture medium as a secretedpolypeptide, although it also may be recovered from host cell lysateswhen directly expressed without a secretory signal. When PCPB isexpressed in a recombinant cell other than one of human origin, the PCPBis completely free of proteins or polypeptides of human origin. However,it is necessary to purify PCPB from recombinant cell proteins orpolypeptides to obtain preparations that are substantially homogeneousas to PCPB. As a first step, the culture medium or lysate is centrifugedto remove particulate cell debris. PCPB thereafter is purified fromcontaminant soluble proteins and polypeptides, for example, byfractionation on immunoaffinity or ion-exchange columns; ethanolprecipitation; reverse phase HPLC; chromatography on silica or on acation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; gel electrophoresis using, for example, SephadexG-75; and chromatography on plasminogen columns to bind the PCPB and onprotein A Sepharose columns to remove contaminants such as IgG.

PCPB variants in which residues have been deleted, inserted orsubstituted are recovered in the same fashion as native PCPB, takingaccount of any substantial changes in properties occasioned by thevariation. For example, preparation of a PCPB fusion with anotherprotein or polypeptide, e.g. a bacterial or viral antigen, facilitatespurification because an immunoaffinity column containing antibody to theantigen can be used to adsorb the fusion. Immunoaffinity columns such asa rabbit polyclonal anti-PCPB column can be employed to absorb the PCPBvariant by binding it to at least one remaining immune epitope. Aprotease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) alsomay be useful to inhibit proteolytic degradation during purification,and antibiotics may be included to prevent the growth of adventitiouscontaminants. One skilled in the art will appreciate that purificationmethods suitable for native PCPB may require modification to account forchanges in the character of PCPB or its variants upon expression inrecombinant cell culture.

PCPB also may be entrapped in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization (for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-[methylmethacylate] microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A., Ed., (1980).

PCPB is believed to find use as a hemostatic regulator for clottingblood, i.e., to coagulate blood, and particularly for treating mammals(e.g., animals or humans) in vivo having a blood clotting disorder suchas hemophilia, especially hemophilia A. Hemophilia A is the result ofFactor VIII deficiency, which mainly occurs in males. The disease is amajor inherited bleeding disorder, occurring in about 0.01% of the malepopulation. PCPB may be useful in cases where Factor VIII cannot beemployed, as when the patient develops antibodies to Factor VIII.

PCPB preparations are also useful in generating antibodies, as standardsin assays for PCPB such as by labeling PCPB for use as a standard in aradioimmunoassay, enzyme-linked immunoassay, or radioreceptor assay, inaffinity purification techniques, and in competitive-type receptorbinding assays when labeled with radioiodine, enzymes, fluorophores,spin labels, and the like.

Since it is often difficult to predict in advance the characteristics ofa variant PCPB, it will be appreciated that some screening of therecovered variant will be needed to select the optimal variant. One canscreen for plasminogen binding, enhanced carboxypeptidase activity,enhanced inhibition of plasminogen activation in the presence of aplasminogen activator and fibrinogen, stability in recombinant cellculture or in plasma (e.g. against proteolytic cleavage), possession ofPCPB antagonist activity (e.g., enhancement of plasminogen activation inthe presence of t-PA and fibrinogen), oxidative stability, ability to besecreted in elevated yields, and the like. For example, a change in theimmunological character of the PCPB molecule, such as affinity for agiven antibody, is measured by a competitive-type immunoassay. Thevariant is assayed for changes in the suppression or enhancement of itsenzymatic activity by determining the kinetics of conversion ofplasminogen to plasmin of the candidate mutant using the chromogenicplasmin substrate S-2251 in the presence of fibrinogen fragments andt-PA using the assay as described in the example below. Modifications ofsuch protein or polypeptide properties as redox or thermal stability,hydrophobicity, susceptibility to proteolytic degradation, or thetendency to aggregate with carriers or into multimers are assayed bymethods well known in the art.

Therapeutic formulations of PCPB for treating blood clotting disordersare prepared for storage by mixing PCPB having the desired degree ofpurity with optional physiologically acceptable carriers, excipients, orstabilizers (Remington's Pharmaceutical Sciences, supra), in the form oflyophilized cake or aqueous solutions. Acceptable carriers, excipientsor stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

PCPB to be used for in vivo administration must be sterile. This isreadily accomplished by filtration through sterile filtration membranes,prior to or following lyophilization and reconstitution. PCPB ordinarilywill be stored in lyophilized form.

Therapeutic PCPB compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

PCPB optionally is combined with or administered in concert with otherblood clotting agents including tissue factor and/or Factor VIII and isused with other conventional therapies for blood clotting disorders.

The route of PCPB or PCPB antibody administration is in accord withknown methods, e.g. injection or infusion by intravenous,intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, or intralesional routes, or by sustained release systemsas noted below. PCPB is administered continuously by infusion or bybolus injection. PCPB antibody is administered in the same fashion, orby administration into the blood stream or lymph.

Suitable examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,Biopolymers, 22: 547-556 [1983]), poly (2-hydroxyethyl-methacrylate)(Langer et al., J. Biomed. Mater. Res., 15: 167-277 [1981] and Langer,Chem. Tech., 12: 98-105 [1982]), ethylene vinyl acetate (Langer et al.,supra) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988).Sustained-release PCPB compositions also include liposomally entrappedPCPB. Liposomes containing PCPB are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980);EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patentapplication 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP102,324. Ordinarily the liposomes are of the small (about 200-800Angstroms) unilamelar type in which the lipid content is greater thanabout 30 mol. % cholesterol, the selected proportion being adjusted forthe optimal PCPB therapy.

An effective amount of PCPB to be employed therapeutically will depend,for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.A typical daily dosage might range from about 1 μg/kg to up to 100 mg/kgor more, depending on the factors mentioned above. Typically, theclinician will administer PCPB until a dosage is reached that achievesthe desired degree of clotting. The progress of this therapy is easilymonitored by conventional assays.

Examples of treatment protocols that may be appropriate are those wellknown for Factor VIII, including those described in Hematology 1987,Education Program, American Society of Hematology, Washington, DC (5-8Dec. 1987) and references cited therein; Sultan et al., Nouv. Rev. Fr.Hematol., 28: 85-89 (1986); Brackmann and Egli, Hemophilia, London,Castle House, pp. 113-119 (1981); Brackmann et al., Lancet, 2: 933(1977); White et al., Blood, 62: 141-145 (1983); Van Leeuwen et al., Br.J. Haematol., 64: 291-297 (1986).

Polyclonal antibodies to PCPB generally are raised in animals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of PCPBand an adjuvant. It may be useful to conjugate PCPB or a fragmentcontaining the target amino acid sequence to a protein that isimmunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl₂, or R¹ N═C═NR, where R and R¹are different alkyl groups.

Animals are immunized against the immunogenic conjugates or derivativesby combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freund's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with 1/5 to 1/10 the original amount of conjugate inFreund's complete adjuvant by subcutaneous injection at multiple sites.7 to 14 days later animals are bled and the serum is assayed foranti-PCPB titer. Animals are boosted until the titer plateaus.Preferably, the animal is boosted with the conjugate of the same PCPB,but conjugated to a different protein and/or through a differentcross-linking agent. Conjugates also can be made in recombinant cellculture as protein fusions. Also, aggregating agents such as alum areused to enhance the immune response.

Monoclonal antibodies are prepared by recovering spleen cells fromimmunized animals and immortalizing the cells in conventional fashion,e.g. by fusion with myeloma cells or by Epstein-Barr (EB)-virustransformation and screening for clones expressing the desired antibody.The monoclonal antibody preferably does not cross-react with acarboxypeptidase A, B, E, M, or N, a non-plasma derived carboxypeptidaseB, or a non-mammalian carboxypeptidase.

PCPB antibodies are useful in diagnostic assays for PCPB. The antibodiesare labeled in the same fashion as PCPB described above and/or areimmobilized on an insoluble matrix. In one embodiment of a receptorbinding assay, an antibody composition that binds to all or a selectedplurality of members of the PCPB family is immobilized on an insolublematrix, the test sample is contacted with the immobilized antibodycomposition to adsorb all PCPB family members, and then the immobilizedfamily members are contacted with a plurality of antibodies specific foreach member, each of the antibodies being individually identifiable asspecific for a predetermined family member, as by unique labels such asdiscrete fluorophores or the like. By determining the presence and/oramount of each unique label, the relative proportion and amount of eachfamily member can be determined.

PCPB antibodies also are useful for the affinity purification of PCPBfrom recombinant cell culture or natural sources. PCPB antibodies thatdo not detectably cross-react with other carboxypeptidases such aspancreas, mast cell, and plasma carboxypeptidase A, non-plasma derivedB, or carboxypeptidases N, E, or M or non-mammalian carboxypeptidasescan be used to purify PCPB free from these other carboxypeptidases.

Suitable diagnostic assays for PCPB and its antibodies are well knownper se. In addition to the bioassays described above and in the exampleswherein the candidate PCPB is tested to see if it inhibits plasminactivity and the activation of plasminogen in the presence of t-PA andfibrinogen, competitive, sandwich and steric inhibition immunoassaytechniques are useful. The competitive and sandwich methods employ aphase-separation step as an integral part of the method while stericinhibition assays are conducted in a single reaction mixture.Fundamentally, the same procedures are used for the assay of PCPB andfor substances that bind PCPB, although certain methods will be favoreddepending upon the molecular weight of the substance being assayed.Therefore, the substance to be tested is referred to herein as ananalyte, irrespective of its status otherwise as an antigen or antibody,and proteins that bind to the analyte are denominated binding partners,whether they be antibodies, cell surface receptors, or antigens.

Analytical methods for PCPB or its antibodies all use one or more of thefollowing reagents: labeled analyte analogue, immobilized analyteanalogue, labeled binding partner, immobilized binding partner andsteric conjugates. The labeled reagents also are known as "tracers."

The label used (and this is also useful to label PCPB nucleic acid foruse as a probe) is any detectable functionality that does not interferewith the binding of analyte and its binding partner. Numerous labels areknown for use in immunoassay, examples including moieties that may bedetected directly, such as fluorochrome, chemiluminscent, andradioactive labels, as well as moieties, such as enzymes, that must bereacted or derivatized to be detected. Examples of such labels includethe radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I, fluorophores such asrare earth chelates or fluorescein and its derivatives, rhodamine andits derivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

Conventional methods are available to bind these labels covalently toproteins or polypeptides. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. Nos. 3,940,475 (fluorimetry) and 3,645,090(enzymes); Hunter et al., Nature, 144: 945 (1962); David et al.,Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol. Methods,40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407-412(1982). Preferred labels herein are enzymes such as horseradishperoxidase and alkaline phosphatase.

The conjugation of such label, including the enzymes, to the antibody isa standard manipulative procedure for one of ordinary skill inimmunoassay techniques. See, for example, O'Sullivan et al., "Methodsfor the Preparation of Enzyme-antibody Conjugates for Use in EnzymeImmunoassay," in Methods in Enzymology, ed. J. J. Langone and H. VanVunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166.Such bonding methods are suitable for use with PCPB or its antibodies,all of which are proteinaceous.

Immobilization of reagents is required for certain assay methods.Immobilization entails separating the binding partner from any analytethat remains free in solution. This conventionally is accomplished byeither insolubilizing the binding partner or analyte analogue before theassay procedure, as by adsorption to a water-insoluble matrix or surface(Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling (forexample, using glutaraldehyde cross-linking), or by insolubilizing thepartner or analogue afterward, e.g., by immunoprecipitation.

Other assay methods, known as competitive or sandwich assays, are wellestablished and widely used in the commercial diagnostics industry.

Competitive assays rely on the ability of a tracer analogue to competewith the test sample analyte for a limited number of binding sites on acommon binding partner. The binding partner generally is insolubilizedbefore or after the competition and then the tracer and analyte bound tothe binding partner are separated from the unbound tracer and analyte.This separation is accomplished by decanting (where the binding partnerwas preinsolubilized) or by centrifuging (where the binding partner wasprecipitated after the competitive reaction). The amount of test sampleanalyte is inversely proportional to the amount of bound tracer asmeasured by the amount of marker substance. Dose-response curves withknown amounts of analyte are prepared and compared with the test resultsto quantitatively determine the amount of analyte present in the testsample. These assays are called ELISA systems when enzymes are used asthe detectable markers.

Another species of competitive assay, called a "homogeneous" assay, doesnot require a phase separation. Here, a conjugate of an enzyme with theanalyte is prepared and used such that when anti-analyte binds to theanalyte the presence of the anti-analyte modifies the enzyme activity.In this case, PCPB or its immunologically active fragments areconjugated with a bifunctional organic bridge to an enzyme such asperoxidase. Conjugates are selected for use with anti-PCPB so thatbinding of the anti-PCPB inhibits or potentiates the enzyme activity ofthe label. This method per se is widely practiced under the name ofEMIT.

Steric conjugates are used in steric hindrance methods for homogeneousassay. These conjugates are synthesized by covalently linking alow-molecular-weight hapten to a small analyte so that antibody tohapten substantially is unable to bind the conjugate at the same time asanti-analyte. Under this assay procedure the analyte present in the testsample will bind anti-analyte, thereby allowing anti-hapten to bind theconjugate, resulting in a change in the character of the conjugatehapten, e.g., a change in fluorescence when the hapten is a fluorophore.

Sandwich assays particularly are useful for the determination of PCPB orPCPB antibodies. In sequential sandwich assays an immobilized bindingpartner is used to adsorb test sample analyte, the test sample isremoved as by washing, the bound analyte is used to adsorb labeledbinding partner, and bound material is then separated from residualtracer. The amount of bound tracer is directly proportional to testsample analyte. In "simultaneous" sandwich assays the test sample is notseparated before adding the labeled binding partner. A sequentialsandwich assay using an anti-PCPB monoclonal antibody as one antibodyand a polyclonal anti-PCPB antibody as the other is useful in testingsamples for PCPB activity.

The foregoing are merely exemplary diagnostic assays for PCPB andantibodies. Other methods now or hereafter developed for thedetermination of these analytes are included within the scope hereof,including the bioassays described above.

The following examples are offered by way of illustration and not by wayof limitation. All literature references cited in the example sectionare expressly incorporated herein by reference.

EXAMPLE I Purification of PCPB

A total of 20 units of human plasma was batch separated with 1 liter oflysine-Sepharose (Pharmacia) pre-equilibrated with phosphate bufferedsaline (PBS). After 2 hours at 4° C. the resin was removed bycentrifugation. The plasminogen-depleted plasma was made 0.8M withammonium sulfate and subsequently centrifuged at 10,000×g for 30 min.The pellet was discarded and the supernatant was made 2.7M ammoniumsulfate and re-centrifuged. The supernatant was discarded and the 2.7Mpellet was dissolved in PBS and extensively dialyzed against the same.

After dialysis, the 2.7M pellet was chromatographed on a plasminogenaffinity column, prepared by coupling plasminogen to glycerol-coatedcontrol pore glass at 15 mg/g as described by Roy et al., J.Chromatography, 303: 225-228 (1984). The column was washed with 10column volumes of PBS and eluted with PBS containing 0.2M ofepsilon-aminocaproic acid (EACA). Fractions containing PCPB (identifiedby SDS-PAGE and amino acid sequence) were pooled, made 1M NaCl, andpassed over a protein-A Sepharose column (Pharmacia) equilibrated in PBSto remove contaminating IgG. Fractions containing PCPB identified asdescribed above were pooled and re-chromatographed on the plasminogenaffinity column. The column was washed with 10 column volumes of PBS andeluted with a 0-50 mM gradient of EACA and the fractions containing PCPB(identified as described above) were recovered.

SDS-PAGE of PCPB before and after protein-A Sepharose purificationrevealed a band at a molecular weight of about 60 kD on both a reducingand non-reducing gel. Sequencing by Edman degradation of the polypeptidebefore protein-A Sepharose purification gave the following N-terminalsequence:

    ______________________________________                                        PheGlnSerGlyGlnValLeuAlaAlaLeuProArgThrSerArgGln-                             ValGlnValLeuGlnAsnLeuThrThrThrTyrGluIsoValLeuArgGlu-                          ProValThrAla.                                                                 (Sequence ID No. 1)                                                           ______________________________________                                    

The purified PCPB was tested to determine its effect on plasminogenactivation. The plasmin-specific substrateH-D-valyl-H-leucyl-H-lysine-paranitroanilide (S-2251) was used in atwo-stage assay to measure the ability of the sample to activateplasminogen. Human fibrinogen (Calbiochem) was made plasminogen free byapplying it to a lysine-Sepharose column and collecting theflow-through. The fibrinogen was used as a stimulator by incubating 450nM of the PCPB sample with 1800 nM fibrinogen and 815 nM Glu-plasminogensolution (commercially available) in 50 mM TrisOH, pH 7.5, buffercontaining 0.15M NaCl and 0.01% Tween 20 (TBST) at a final volume of 0.3ml for 5 minutes at 37° C. Subsequently, 0.1 ml of TBST containing 25 ngof human rt-PA (Activase® brand alteplase, Genentech, Inc.) was addedand the mixture was incubated another 10 minutes at 37° C. Plasmingenerated was then determined by the addition of S-2251 to a finalconcentration of 1 mM. After 10 min. at 37° C. 0.1 ml of 50% glacialacetic acid was added to quench the reaction and absorbance at 405 nmwas determined.

The maximum rate of this reaction was observed in the absence andpresence of fibrinogen that acts as a stimulator of the reaction. Also,control samples were run without the PCPB.

The optical density at 405 nm is shown for these experiments in FIG. 1.It can be seen that PCPB blocks the action of t-PA in convertingplasminogen to plasmin only in the presence of fibrinogen.

The purified PCPB was also tested to determine its effect on plasminactivity. 3.6 nM plasmin (Helena Labs) was incubated with either PCPB(303 nM) or anti-plasmin (American Diagnostic) (256 nM) for 15 minutesat 37° C., and plasmin activity was determined by the S-2251 assaydescribed above. The results, shown in FIG. 2, where "CONT." indicatescontrol (plasmin alone) and "AP" indicates anti-plasmin, demonstratethat PCPB does not block the action of plasmin.

EXAMPLE II Cloning and Expression of PCPB DNA

Cloning:

Attempts to identify and isolate DNA encoding PCPB from a human livercDNA library using two probes based on the 37 residues of amino-terminalamino acid sequence obtained from plasma-derived PCPB were unsuccessful.Specifically, the initial strategy was to design two longoligonucleotide probes, 45 and 63 bases in length, each representing asingle, non-degenerate sequence encoding a portion of the 37 amino acidsequence [the 45-mer encoded amino acids 16-29 and the 63-mer encodedamino acids 1-19]. These probes were labeled and used to screen a humanliver cDNA library in the vector lambda gt10 [Ullrich et al., Nature,309: 418-425 (1984); Huynh et al., in DNA Cloning, A Practical Approach,ed. Glover, D. (IRL, Oxford), Vol. 1, pp. 49-78 (1985)]. The 63-merhybridized to some 20 clones out of a million at normal stringency, andthe 45-mer hybridized to zero out of a million clones. The clones thathybridized to the 63-mer had long (up to 4.5 kb) inserts, none of whichencoded a protein with the known amino-terminal amino acid sequence ofPCPB. The conclusion from these experiments was that cloning PCPB by thestandard technique of probing cDNA libraries with a single longoligonucleotide was a failure.

Instead, to identify the PCPB gene, it was necessary to amplify humancDNA using PCR (Mullis et al., Cold Spring Harbor Symp. Quant. Biol.,51: 263 [1987]). Three groups of highly degenerate PCR primers weredesigned. All primers were 26 bases in length. Primer pools pbp.8 andpbp.9 were 16,384-fold and 49,152-fold degenerate, respectively, andrepresented all possible coding sequences for the first 9 and last 9amino acids of the 37-amino-acid sequence. Primer pbp.10 was 24,576-folddegenerate, and was internal to pbp.8 and pbp.9. This primer representedall possible coding sequences for amino acids 9-17 of the knownsequence. The expected PCR product from primers pbp.8 and pbp.9 was 105bp, while the expected PCR product from primers pbp.8 and pbp.10 was 95bp in length.

Human mRNA was used as the initial starting material for PCR. RNAextracted from human liver and kidney by known procedures [for example,as described in Section 7.3 of Sambrook et al., supra] was reversetranscribed to cDNA with reverse transcriptase using known techniques asdescribed in Section 8.3-8.53 of Sambrook et al., supra. Bothsingle-stranded and double-stranded cDNA [the latter prepared asdescribed by Sambrook et al. ] was amplified with the degenerate primerpools. The conditions for amplification were as follows:

    ______________________________________                                        denat. 95° C. 5' once initially                                        denat. 95° C. 1'                                                       anneal 50° C. 1'                                                                        30 cycles                                                    extens. 72° C. 1'                                                      extens. 72° C. 15'                                                     10   μl 10× buffer (final = 50 mM KCl, 10 mM Tris pH 8.4, 3.0             mM MgCl.sub.2)                                                           3    μl human cDNA (3 μg)                                               7.5  ng/μl primer pbp.8 (approx. 1 μg = .sup.˜ 2.6 μM of            26 mer,                                                                       therefore 10.sup.3 degen = nM, 10.sup.6 = pM)                            7.5  ng/μl primer pbp.9                                                    7.5  ng/μl primer pbp.10                                                   10   μl 10× dNTPs (final = 0.2 mM dNTPs)                             1    μl Taq polymerase                                                     61   μl dH.sub.2 O                                                         107.5                                                                              μl V.sub.T (total volume)                                             ______________________________________                                    

No PCR products of the correct expected size were produced.

A bi-phasic PCR protocol was then utilized, in which the first 10 cyclesof amplification were carried out at 50° C. annealing temperature andthe subsequent 20 cycles of amplification were carried out at reduced(40° C.) stringency. In addition, the product of the reaction using theoutermost primers (pbp.8 and pbp.9) was subjected to a second round ofbi-phasic PCR using the more internal primers (pbp.8 and pbp.10). Thissecond round of PCR amplification produced, along with a large number ofother PCR products, the expected 95-bp band from liver, but not fromkidney, cDNA.

This 95-bp PCR product was cloned into pUC218 (prepared by ligating thenucleotides TCGAGAGATCTATCGATT into the vector pUCl18 at the locationindicated in FIG. 3) and sequenced. pUC118 is described by Vieira andMessing, Meth. Enzymol., 153: 3-11 (1987). Briefly, pUC118 is a 3.2 kbplasmid with ampicillin resistance and M13 IG region, and a sequenceencoding the lacZ peptide containing unique restriction sites forcloning. pUC118 is pUC18 (Norrander et al., Gene, 26: 101 [1983]) withthe IG region of M13 from the HgiAI site (5465) to the DraI site (5941)inserted at the unique NdeI site (2499) of pUC. The orientation of theM13 IG region is such that the strand of the lac region that is packagedas ssDNA is the same as in the M13mp vectors.

The DNA sequence of the PCR product, which is shown in FIG. 4 (SequenceID No. 2), was capable of encoding the sequenced first 37 amino acids ofPCPB (with the exception of the Trp at position 27, which was in error).Since this PCR product was obtained using highly degenerate primers, theDNA sequence at the ends that contain these primers could not be assumedto be identical to the authentic mRNA sequence for PCPB. Therefore, forfull-length clones, a single 46-bp oligonucleotide probe from theinterior region of this 95-bp sequence was synthesized, having thesequence spanning the nucleotides shown in FIG. 4, i.e. 133 to 178. Thisprobe was radiolabeled using conventional techniques and hybridized athigh stringency [50% formamide, 5×SSC (0.75M NaCl, 0.075M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washing at 42° C. in 0.2×SSC and0.1% SDS] to a human liver cDNA library in the vector lambda gt10 asdescribed above.

A total of 110 positives were obtained from 1.3 million clones (thus,positive clones appeared at a frequency of 0.008%). Five of thesepositive clones, assigned the designation PBP that is equivalent to PCPB[PBP1a, PBP1b, PBP2a, PBP2b, and PBP2c], were purified and grown up, andtheir cDNA inserts were subcloned into pUC218. One subclone, PBP1b(deposited with the ATCC as ATCC No. 40,927), was sequenced asdouble-stranded supercoiled templates, using both universal primers andspecific internal synthesized primers as described in Section 13.70 ofSambrook et al., supra. Portions of other clones were partiallysequenced by the same method, using universal primers.

DNA sequencing revealed that at least 4 out of these 5 clonesrepresented different clones of the same gene. The DNA sequence predictsa single open reading frame 1269 bases long, from which is deduced aprimary translation product 423 amino acids in length. Flanking thisopen reading frame are ca. 150 bp of 5' untranslated sequence and 421 bpof 3' untranslated sequence, which include the polyadenylation signalAATAAA 22 bp upstream from a poly A tail. Following the initiatormethionine, there is a sequence 22 amino acids long that is notcontained in PCPB. This sequence contains several features common toknown signal peptides, including a generally hydrophobic character and aterminal alanine, after which begins the amino-terminal sequence foundin PCPB.

Searches of DNA and protein sequence databases (Genbank and Dayhoff,respectively) reveal that this novel polypeptide has substantialsequence identity with known carboxypeptidases, including rat pancreascarboxypeptidase A1 and A2, rat pancreas carboxypeptidase B, bovinepancreas carboxypeptidase B, and both murine and human mast cellcarboxypeptidase A. The greatest homologies were found withcarboxypeptidases A and B, and a comparison of the sequence ofpreprohPCPB (Sequence ID No. 3) with the known sequences of prepro-ratcarboxypeptidase B (Sequence ID No. 4), prepro-rat carboxypeptidase A1(Sequence ID No. 5), prepro-human mast cell carboxypeptidase A (SequenceID No. 6), prepro-mouse mast cell carboxypeptidase A (Sequence ID No.7), and prepro-rat carboxypeptidase A2 (Sequence ID No. 8) is shown inFIG. 5. Overall, there is about 40% sequence identity at the amino acidlevel between preprohPCPB and prepro-human mast cell carboxypeptidase A,and between preproPCPB and prepro-rat carboxypeptidase B.

In particular, there is identity at three of four amino acids that formthe catalytic site, at five of seven amino acids that form the substratebinding pocket, and at four cysteine residues known to formintramolecular disulfide bonds in two pairs in mast cellcarboxypeptidase A (Reynolds et al., J. Biol. Chem., 264: 20094-99[1989]).

Human PCPB shares a small amount of sequence identity with human plasmacarboxypeptidases N, M, and E (e.g., positions 232 to 237 of maturehPCPB correspond to positions 138-143 of the human carboxypeptidase N48-55 kD subunit whose sequence is shown in FIG. 6 of Tan et al., J.Biol. Chem., supra, spanning from Asp to Phe). In addition, human PCPBhas the same substrate binding sites as, and shares a fifth and sixthcysteine residue with, bovine and rat carboxypeptidase B. Thesecysteines form a third intramolecular disulfide bond that is not presentin human mast cell carboxypeptidase A. Also, the serine cleavage sitethat is cleaved to form proteolytically active rat cell carboxypeptidaseB when activated by trypsin also exists in PCPB in an analogous position(Arg residue at position 92). All of these features strongly suggestthat PCPB is a functional carboxypeptidase. Because hPCPB has the sameamino acid (aspartic acid at position 348) at the region incarboxypeptidases that determines substrate specificity ascarboxypeptidase B (Asp), it is believed that PCPB represents aplasma-derived carboxypeptidase B.

Expression:

The following protocol for expressing PCPB DNA and purifying theresultant PCPB is expected to provide sufficient PCPB for assaypurposes.

A cytomegalovirus-based expression vector called pRK5, described inGorman et al., DNA and Protein Engineering Techniques, 2: 1 (1990) andin EP 307,247 published 15 Mar. 1989, was employed as the expressionvector. The PCPB cDNA insert from clone PBP.1b was cut from pUC218 inwhich it was cloned using partial EcoRI digest to obtain the ⁻ 1.7 kbinsert fragment. This DNA fragment was then ligated into pRK5 previouslycut with EcoRI to accommodate the DNA fragment using standard ligationmethodology as described in Sections 5.10 to 5.11 of Sambrook et al.,supra. The resulting vector was called pRK-5hPBP.1b.

Human embryonic kidney 293 cells (Graham et al., J. Gen. Virol., 36: 59[1977], subclone 293TSA transfected with the temperature-sensitive largeT-antigen gene) were grown to 70% confluence in 6-well plates in aDMEM:F12 (1:1) medium containing 1 mM HEPES buffer, 0.29 g/l glutamine,2.44 g/l sodium bicarbonate, 0.55 g/l sodium pyruvate, pH 6.95,supplemented with 10% whole fetal calf serum. The day before thetransfection the cells were counted, the medium was aspirated off, andthe cells were trypsinized and resuspended in the same DMEM:F12(1:1)-based medium containing 10% whole fetal calf serum that had beenrun through a lysine-containing column to remove plasminogen. Then thesuspension was adjusted to 266,000 cells/ml, seeded at 3 ml per well ofa six-well plate (800,000 cells/well), and incubated until the day ofthe transfection.

A total of 5 μg of the plasmid DNA (pRK-ShPBP.1b) was dissolved in 150μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227M CaCl₂. Added to this (dropwisewhile vortexing) was 150 μl of 50 mM HEPES buffer (pH 7.35), 280 mMNaCl, 1.5 mM NaPO₄, and the precipitate was allowed to form for ten min.at 25° C. The suspended precipitate was then added to the cells in the60-mm tissue culture plate and allowed to settle overnight in theincubator. The medium was then aspirated off and replaced with DMEM:F12(1:1)-based serum-free medium called PS-04 containing insulin,transferrin, trace elements, and lipids and described in U.S. Ser. No.07/592,141, supra.

After the cells were incubated for four hours in the presence of 200μCi/ml ³⁵ S-cysteine and 200 μCi ³⁵ S-methionine, conditioned medium wasthen collected, concentrated 5-fold by lyophilization, and loaded on a15% SDS gel, which was subsequently enhanced, dried, and exposed to filmfor two hours. Because of an interfering protein, it could not bedetermined from the gel whether a polypeptide of approximately theexpected size (60 kD) was obtained.

It is expected that a polypeptide of the correct molecular size would bedetected if the transfection medium were placed on a plasminogenaffinity column as described above washed with 10 column volumes of PBSand eluted with a 0 to 50 mM EACA gradient to remove the interferingcontaminant, followed preferably by protein A-Sepharose columnchromatography as described above.

Large-scale expression of PCPB is performed by transiently introducingby the dextran sulfate method (Sompayrac and Danna, Proc. Natl. Acad.Sci. USA, 12: 7575 [1981]) 700 μg of pRK-5hPBP.1b into the humanembryonal kidney 293 cell line grown to maximal density (1.5 liters) ina 3-liter Belco microcarrier spinner flask. The cells are firstconcentrated from the spinner flask by centrifugation, and washed withPBS, and the DNA-dextran precipitate is incubated on the cell pellet forfour hours. The cells are treated with 20% glycerol for 90 seconds,washed with a medium such as 50:50 DMEM:F-12 medium, and re-introducedinto a 3-liter spinner flask containing 1.5 liter of the above mediumplus 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. The aboveprotocol is performed for three separate 3-liter cultures.

In a different expression protocol, pRK-5hPBP.1b was transfected intoCOS cells using the same conditions as for the 293 cells describedabove.

For larger-scale production of PCPB, the preferred vector is aSV40-driven vector such as pSVI6B described above, and the preferredhost cells are Chinese hamster ovary cells.

Deposit of Materials

The following plasmid DNA has been deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC):

    ______________________________________                                        Plasmid DNA ATCC Accession No.                                                                             Deposit Date                                     ______________________________________                                        pPBP.1b     40,927           29 Nov. 1990                                     ______________________________________                                    

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable deposit for 30 years fromthe date of deposit. The plasmid DNA will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the plasmid DNA to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the plasmid DNA to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC §122 and the Commissioner's rules pursuant thereto (including37 CFR §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the depositedDNA should be lost or destroyed when transformed into a suitable hostcultivated under suitable conditions, it will be promptly replaced onnotification with a specimen of the same DNA. Availability of thedeposited DNA is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the plasmid DNA deposited,since the deposited embodiment is intended as a single illustration ofone aspect of the invention and any constructs that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial herein does not constitute an admission that the writtendescription herein contained is inadequate to enable the practice of anyaspect of the invention, including the best mode thereof, nor is it tobe construed as limiting the scope of the claims to the specificillustration that it represents. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       PheGlnSerGlyGlnValLeuAlaAlaLeuProArgThrSerArg                                 151015                                                                        GlnValGlnValLeuGlnAsnLeuThrThrThrTyrGluIleVal                                 202530                                                                        LeuArgGluProValThrAla                                                         3537                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1749 bases                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AGCTCGTCGACCTTTCTCTGAAGAGAAAATTGCTGTTGGGATGAAG46                              MetLys                                                                        22                                                                            CTTTGCAGCCTTGCAGTCCTTGTACCCATTGTTCTCTTC85                                     LeuCysSerLeuAlaValLeuValProIleValLeuPhe                                       20-15-10                                                                      TGTGAGCAGCATGTCTTCGCGTTTCAGAGTGGCCAAGTT124                                    CysGluGlnHisValPheAlaPheGlnSerGlyGlnVal                                       515                                                                           CTAGCTGCTCTTCCTAGAACCTCTAGGCAAGTTCAAGTT163                                    LeuAlaAlaLeuProArgThrSerArgGlnValGlnVal                                       1015                                                                          CTACAGAATCTTACTACAACATATGAGATTGTTCTCTGG202                                    LeuGlnAsnLeuThrThrThrTyrGluIleValLeuTrp                                       202530                                                                        CAGCCGGTAACAGCTGACCTTATTGTGAAGAAAAAACAA241                                    GlnProValThrAlaAspLeuIleValLysLysLysGln                                       354045                                                                        GTCCATTTTTTTGTAAATGCATCTGATGTCGACAATGTG280                                    ValHisPhePheValAsnAlaSerAspValAspAsnVal                                       5055                                                                          AAAGCCCATTTAAATGTGAGCGGAATTCCATGCAGTGTC319                                    LysAlaHisLeuAsnValSerGlyIleProCysSerVal                                       606570                                                                        TTGCTGGCAGACGTGGAAGATCTTATTCAACAGCAGATT358                                    LeuLeuAlaAspValGluAspLeuIleGlnGlnGlnIle                                       7580                                                                          TCCAACGACACAGTCAGCCCCCGAGCCTCCGCATCGTAC397                                    SerAsnAspThrValSerProArgAlaSerAlaSerTyr                                       859095                                                                        TATGAACAGTATCACTCACTAAATGAAATCTATTCTTGG436                                    TyrGluGlnTyrHisSerLeuAsnGluIleTyrSerTrp                                       100105110                                                                     ATAGAATTTATAACTGAGAGGCATCCTGATATGCTTACA475                                    IleGluPheIleThrGluArgHisProAspMetLeuThr                                       115120                                                                        AAAATCCACATTGGATCCTCATTTGAGAAGTACCCACTC514                                    LysIleHisIleGlySerSerPheGluLysTyrProLeu                                       125130135                                                                     TATGTTTTAAAGGTTTCTGGAAAAGAACAAACAGCCAAA553                                    TyrValLeuLysValSerGlyLysGluGlnThrAlaLys                                       140145                                                                        AATGCCATATGGATTGACTGTGGAATCCATGCCAGAGAA592                                    AsnAlaIleTrpIleAspCysGlyIleHisAlaArgGlu                                       150155160                                                                     TGGATCTCTCCTGCTTTCTGCTTGTGGTTCATAGGCCAT631                                    TrpIleSerProAlaPheCysLeuTrpPheIleGlyHis                                       165170175                                                                     ATAACTCAATTCTATGGGATAATAGGGCAATATACCAAT670                                    IleThrGlnPheTyrGlyIleIleGlyGlnTyrThrAsn                                       180185                                                                        CTCCTGAGGCTTGTGGATTTCTATGTTATGCCGGTGGTT709                                    LeuLeuArgLeuValAspPheTyrValMetProValVal                                       190195200                                                                     AATGTGGACGGTTATGACTACTCATGGAAAAAGAATCGA748                                    AsnValAspGlyTyrAspTyrSerTrpLysLysAsnArg                                       205210                                                                        ATGTGGAGAAAGAACCGTTCTTTCTATGCGAACAATCAT787                                    MetTrpArgLysAsnArgSerPheTyrAlaAsnAsnHis                                       215220225                                                                     TGCATCGGAACAGACCTGAATAGGAACTTTGCTTCCAAA826                                    CysIleGlyThrAspLeuAsnArgAsnPheAlaSerLys                                       230235240                                                                     CACTGGTGTGAGGAAGGTGCATCCAGTTCCTCATGCTCG865                                    HisTrpCysGluGluGlyAlaSerSerSerSerCysSer                                       245250                                                                        GAAACCTACTGTGGACTTTATCCTGAGTCAGAACCAGAA904                                    GluThrTyrCysGlyLeuTyrProGluSerGluProGlu                                       255260265                                                                     GTGAAGGCAGTGGCTAGTTTCTTGAGAAGAAATATCAAC943                                    ValLysAlaValAlaSerPheLeuArgArgAsnIleAsn                                       270275                                                                        CAGATTAAAGCATACATCAGCATGCATTCATACTCCCAG982                                    GlnIleLysAlaTyrIleSerMetHisSerTyrSerGln                                       280285290                                                                     CATATAGTGTTTCCATATTCCTATACACGAAGTAAAAGC1021                                   HisIleValPheProTyrSerTyrThrArgSerLysSer                                       295300305                                                                     AAAGACCATGAGGAACTGTCTCTAGTAGCCAGTGAAGCA1060                                   LysAspHisGluGluLeuSerLeuValAlaSerGluAla                                       310315                                                                        GTTCGTGCTATTGAGAAAACTAGTAAAAATACCAGGTAT1099                                   ValArgAlaIleGluLysThrSerLysAsnThrArgTyr                                       320325330                                                                     ACACATGGCCATGGCTCAGAAACCTTATACCTAGCTCCT1138                                   ThrHisGlyHisGlySerGluThrLeuTyrLeuAlaPro                                       335340                                                                        GGAGGTGGGGACGATTGGATCTATGATTTGGGCATCAAA1177                                   GlyGlyGlyAspAspTrpIleTyrAspLeuGlyIleLys                                       345350355                                                                     TATTCGTTTACAATTGAACTTCGAGATACGGGCACATAC1216                                   TyrSerPheThrIleGluLeuArgAspThrGlyThrTyr                                       360365370                                                                     GGATTCTTGCTGCCGGAGCGTTACATCAAACCCACCTGT1255                                   GlyPheLeuLeuProGluArgTyrIleLysProThrCys                                       375380                                                                        AGAGAAGCTTTTGCCGCTGTCTCTAAAATAGCTTGGCAT1294                                   ArgGluAlaPheAlaAlaValSerLysIleAlaTrpHis                                       385390395                                                                     GTCATTAGGAATGTTTAATGCCCCTGATTTTATCATTCTGCTTCCG1340                            ValIleArgAsnVal                                                               400401                                                                        TATTTTAATTTACTGATTCCAGCAAGACCAAATCATTGTATCAGATTATT1390                        TTTAAGTTTTATCCGTAGTTTTGATAAAAGATTTTCCTATTCCTTGGTTC1440                        TGTCAGAGAACCTAATAAGTGCTACTTTGCCATTAAGGCAGACTAGGGTT1490                        CATGTCTTTTTACCCTTTAAAAAAAAATTGTAAAAGTCTAGTTACCTACT1540                        TTTTCTTTGATTTTCGACGTTTGACTAGCCATCTCAAGCAACTTTCGACG1590                        TTTGACTAGCCATCTCAAGCAAGTTTAATCAAAGATCATCTCACGCTGAT1640                        CATTGGATCCTACTCAACAAAAGGAAGGGTGGTCAGAAGTACATTAAAGA1690                        TTTCTGCTCCAAATTTTCAATAAATTTCTTCTTCTCCTTTAAAAAAAAAA1740                        AAAAAAAAA1749                                                                 (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 423 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetLysLeuCysSerLeuAlaValLeuValProIleValLeuPhe                                 151015                                                                        CysGluGlnHisValPheAlaPheGlnSerGlyGlnValLeuAla                                 202530                                                                        AlaLeuProArgThrSerArgGlnValGlnValLeuGlnAsnLeu                                 354045                                                                        ThrThrThrTyrGluIleValLeuTrpGlnProValThrAlaAsp                                 505560                                                                        LeuIleValLysLysLysGlnValHisPhePheValAsnAlaSer                                 657075                                                                        AspValAspAsnValLysAlaHisLeuAsnValSerGlyIlePro                                 808590                                                                        CysSerValLeuLeuAlaAspValGluAspLeuIleGlnGlnGln                                 95100105                                                                      IleSerAsnAspThrValSerProArgAlaSerAlaSerTyrTyr                                 110115120                                                                     GluGlnTyrHisSerLeuAsnGluIleTyrSerTrpIleGluPhe                                 125130135                                                                     IleThrGluArgHisProAspMetLeuThrLysIleHisIleGly                                 140145150                                                                     SerSerPheGluLysTyrProLeuTyrValLeuLysValSerGly                                 155160165                                                                     LysGluGlnThrAlaLysAsnAlaIleTrpIleAspCysGlyIle                                 170175180                                                                     HisAlaArgGluTrpIleSerProAlaPheCysLeuTrpPheIle                                 185190195                                                                     GlyHisIleThrGlnPheTyrGlyIleIleGlyGlnTyrThrAsn                                 200205210                                                                     LeuLeuArgLeuValAspPheTyrValMetProValValAsnVal                                 215220225                                                                     AspGlyTyrAspTyrSerTrpLysLysAsnArgMetTrpArgLys                                 230235240                                                                     AsnArgSerPheTyrAlaAsnAsnHisCysIleGlyThrAspLeu                                 245250255                                                                     AsnArgAsnPheAlaSerLysHisTrpCysGluGluGlyAlaSer                                 260265270                                                                     SerSerSerCysSerGluThrTyrCysGlyLeuTyrProGluSer                                 275280285                                                                     GluProGluValLysAlaValAlaSerPheLeuArgArgAsnIle                                 290295300                                                                     AsnGlnIleLysAlaTyrIleSerMetHisSerTyrSerGlnHis                                 305310315                                                                     IleValPheProTyrSerTyrThrArgSerLysSerLysAspHis                                 320325330                                                                     GluGluLeuSerLeuValAlaSerGluAlaValArgAlaIleGlu                                 335340345                                                                     LysThrSerLysAsnThrArgTyrThrHisGlyHisGlySerGlu                                 350355360                                                                     ThrLeuTyrLeuAlaProGlyGlyGlyAspAspTrpIleTyrAsp                                 365370375                                                                     LeuGlyIleLysTyrSerPheThrIleGluLeuArgAspThrGly                                 380385390                                                                     ThrTyrGlyPheLeuLeuProGluArgTyrIleLysProThrCys                                 395400405                                                                     ArgGluAlaPheAlaAlaValSerLysIleAlaTrpHisValIle                                 410415420                                                                     ArgAsnVal                                                                     423                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 396 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetLeuLeuLeuLeuAlaLeuValSerValAlaLeuAlaHisAla                                 151015                                                                        SerGluGluHisPheAspAsnArgValTyrArgValSerValHis                                 202530                                                                        GlyGluAspHisValAsnLeuIleGlnGluLeuAlaAsnThrLys                                 354045                                                                        GluIleAspPheTrpLysProAspSerAlaThrGlnValLysPro                                 505560                                                                        LeuThrThrValAsnGluValHisThrGluValLeuIleSerAsn                                 657075                                                                        ValArgAsnAlaLeuGluSerGlnPheAspSerHisThrArgAla                                 808590                                                                        SerGlyHisSerThrThrLysThrAsnLysTrpGluThrIleGlu                                 95100105                                                                      AlaTrpIleGlnGlnValAlaThrAspAsnProAspLeuValThr                                 110115120                                                                     GlnSerValIleGlyThrThrPheGluGlyArgAsnMetTyrVal                                 125130135                                                                     LeuLysIleGlyLysThrArgProAsnLysProAlaIlePheIle                                 140145150                                                                     AspCysGlyPheHisAlaArgGluTrpIleSerProAlaPheCys                                 155160165                                                                     GlnTrpPheAlaArgGluAlaValArgThrTyrAsnGlnGluIle                                 170175180                                                                     HisMetLysGlnLeuLeuAspGluLeuAspPheTyrValLeuPro                                 185190195                                                                     ValValAsnIleAspGlyTyrValTyrThrTrpThrLysAspArg                                 200205210                                                                     MetTrpArgLysThrArgSerThrMetAlaGlySerSerCysLeu                                 215220225                                                                     GlyValArgProAsnArgAsnPheAsnAlaGlyTrpCysGluVal                                 230235240                                                                     GlyAlaSerArgSerProCysSerGluThrThrCysGlyProAla                                 245250255                                                                     ProGluSerGluLysGluThrLysAlaLeuAlaAspPheIleArg                                 260265270                                                                     AsnAsnLeuSerThrIleLysAlaThrLeuThrIleHisSerTyr                                 275280285                                                                     SerGlnMetMetLeuTyrProTyrSerTyrAspTyrLysLeuPro                                 290295300                                                                     GluAsnTyrGluGluLeuAsnAlaLeuValLysGlyAlaAlaLys                                 305310315                                                                     GluLeuAlaThrLeuHisGlyThrLysTyrThrTyrGluProGly                                 320325330                                                                     AlaThrThrIleTyrProAlaAlaGlyGlySerAspAspTrpSer                                 335340345                                                                     TyrAspGlnGlyIleLysTyrSerPheThrPheGluLeuArgAsp                                 350355360                                                                     ThrGlyPhePheGlyPheLeuLeuProGluSerGlnIleArgGln                                 365370375                                                                     ThrCysGluGluThrMetLeuAlaValLysTyrIleAlaAsnTyr                                 380385390                                                                     ValArgGluHisLeuTyr                                                            395396                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 419 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetLysArgLeuLeuIleLeuSerLeuLeuLeuGluAlaValCys                                 151015                                                                        GlyAsnGluAsnPheValGlyHisGlnValLeuArgIleSerAla                                 202530                                                                        AlaAspGluAlaGlnValGlnLysValLysGluLeuGluAspLeu                                 354045                                                                        GluHisLeuGlnLeuAspPheTrpArgAspAlaAlaArgAlaGly                                 505560                                                                        IleProIleAspValArgValProPheProSerIleGlnSerVal                                 657075                                                                        LysAlaPheLeuGluTyrHisGlyIleSerTyrGluIleMetIle                                 808590                                                                        GluAspValGlnLeuLeuLeuAspGluGluLysGlnGlnMetSer                                 95100105                                                                      AlaPheGlnAlaArgAlaLeuSerThrAspSerPheAsnTyrAla                                 110115120                                                                     ThrTyrHisThrLeuAspGluIleTyrGluPheMetAspLeuLeu                                 125130135                                                                     ValAlaGluHisProGlnLeuValSerLysIleGlnIleGlyAsn                                 140145150                                                                     ThrPheGluGlyArgProIleHisValLeuLysPheSerThrGly                                 155160165                                                                     GlyThrAsnArgProAlaIleTrpIleAspThrGlyIleHisSer                                 170175180                                                                     ArgGluTrpValThrGlnAlaSerGlyValTrpPheAlaLysLys                                 185190195                                                                     ValThrLysAspTyrGlyGlnAspProThrPheThrAlaValLeu                                 200205210                                                                     AspAsnMetAspIlePheLeuGluIleValThrAsnProAspGly                                 215220225                                                                     PheAlaTyrThrHisLysThrAsnArgMetTrpArgLysThrArg                                 230235240                                                                     SerHisThrGlnGlySerLeuCysValGlyValAspProAsnArg                                 245250255                                                                     AsnTrpAspAlaGlyLeuGlyLysAlaGlyAlaSerSerAsnPro                                 260265270                                                                     CysSerGluThrTyrArgGlyLysPheProAsnSerGluValGlu                                 275280285                                                                     ValLysSerIleValAspPheValThrSerHisGlyAsnIleLys                                 290295300                                                                     AlaPheIleSerIleHisSerTyrSerGlnLeuLeuLeuTyrPro                                 305310315                                                                     TyrGlyTyrThrSerGluProAlaProAspGlnAlaGluLeuAsp                                 320325330                                                                     GlnLeuAlaLysSerAlaValThrAlaLeuThrSerLeuHisGly                                 335340345                                                                     ThrGluPheLysTyrGlySerIleIleAspThrIleTyrGlnAla                                 350355360                                                                     SerGlySerThrIleAspTrpThrTyrSerGlnGlyIleLysTyr                                 365370375                                                                     SerPheThrPheGluLeuArgAspThrGlyLeuArgGlyPheLeu                                 380385390                                                                     LeuProAlaSerGlnIleIleProThrAlaGluGluThrTrpLeu                                 395400405                                                                     AlaLeuLeuThrIleMetAspHisThrValLysHisProTyr                                    410415419                                                                     (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 417 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetArgLeuIleLeuProValGlyLeuIleAlaThrThrLeuAla                                 151015                                                                        IleAlaProValArgPheAspArgGluLysValPheArgValLys                                 202530                                                                        ProGlnAspGluLysGlnAlaAspIleIleLysAspLeuAlaLys                                 354045                                                                        ThrAsnGluLeuAspPheTrpTyrProGlyAlaThrHisHisVal                                 505560                                                                        AlaAlaAsnMetMetValAspPheArgValSerGluLysGluSer                                 657075                                                                        GlnAlaIleGlnSerAlaLeuAspGlnAsnLysMetHisTyrGlu                                 808590                                                                        IleLeuIleHisAspLeuGlnGluGluIleGluLysGlnPheAsp                                 95100105                                                                      ValLysGluAspIleProGlyArgHisSerTyrAlaLysTyrAsn                                 110115120                                                                     AsnTrpGluLysIleValAlaTrpThrGluLysMetMetAspLys                                 125130135                                                                     TyrProGluMetValSerArgIleLysIleGlySerThrValGlu                                 140145150                                                                     AspAsnProLeuTyrValLeuLysIleGlyGluLysAsnGluArg                                 155160165                                                                     ArgLysAlaIlePheMetAspCysGlyIleHisAlaArgGluTrp                                 170175180                                                                     ValSerProAlaPheCysGlnTrpPheValTyrGlnAlaThrLys                                 185190195                                                                     ThrTyrGlyArgAsnLysIleMetThrLysLeuLeuAspArgMet                                 200205210                                                                     AsnPheTyrIleLeuProValPheAsnValAspGlyTyrIleTrp                                 215220225                                                                     SerTrpThrLysAsnArgMetTrpArgLysAsnArgSerLysAsn                                 230235240                                                                     GlnAsnSerLysCysIleGlyThrAspLeuAsnArgAsnPheAsn                                 245250255                                                                     AlaSerTrpAsnSerIleProAsnThrAsnAspProCysAlaAsp                                 260265270                                                                     AsnTyrArgGlySerAlaProGluSerGluLysGluThrLysAla                                 275280285                                                                     ValThrAsnPheIleArgSerHisLeuAsnGluIleLysValTyr                                 290295300                                                                     IleThrPheHisSerTyrSerGlnMetLeuLeuPheProTyrGly                                 305310315                                                                     TyrThrSerLysLeuProProAsnHisGluAspLeuAlaLysVal                                 320325330                                                                     AlaLysIleGlyThrAspValLeuSerThrArgTyrGluThrArg                                 335340345                                                                     TyrIleTyrGlyProIleGluSerThrIleTyrProIleSerGly                                 350355360                                                                     SerSerLeuAspTrpAlaTyrAspLeuGlyIleLysHisThrPhe                                 365370375                                                                     AlaPheGluLeuArgAspLysGlyLysPheGlyPheLeuLeuPro                                 380385390                                                                     GluSerArgIleLysProThrCysArgGluThrMetLeuAlaVal                                 395400405                                                                     LysPheIleAlaLysTyrIleLeuLysHisThrSer                                          410415417                                                                     (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 417 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetArgPhePheLeuLeuMetAlaValIleTyrThrThrLeuAla                                 151015                                                                        IleAlaProValHisPheAspArgGluLysValPheArgValLys                                 202530                                                                        LeuGlnAsnGluLysHisAlaSerValLeuLysAsnLeuThrGln                                 354045                                                                        SerIleGluLeuAspPheTrpTyrProAspAlaIleHisAspIle                                 505560                                                                        AlaValAsnMetThrValAspPheArgValSerGluLysGluSer                                 657075                                                                        GlnThrIleGlnSerThrLeuGluGlnHisLysIleHisTyrGlu                                 808590                                                                        IleLeuIleHisAspLeuGlnGluGluIleGluLysGlnPheAsp                                 95100105                                                                      ValLysAspGluIleAlaGlyArgHisSerTyrAlaLysTyrAsn                                 110115120                                                                     AspTrpAspLysIleValSerTrpThrGluLysMetLeuGluLys                                 125130135                                                                     HisProGluMetValSerArgIleLysIleGlySerThrValGlu                                 140145150                                                                     AspAsnProLeuTyrValLeuLysIleGlyLysLysAspGlyGlu                                 155160165                                                                     ArgLysAlaIlePheMetAspCysGlyIleHisAlaArgGluTrp                                 170175180                                                                     IleSerProAlaPheCysGlnTrpPheValTyrGlnAlaThrLys                                 185190195                                                                     SerTyrGlyLysAsnLysIleMetThrLysLeuLeuAspArgMet                                 200205210                                                                     AsnPheTyrValLeuProValPheAsnValAspGlyTyrIleTrp                                 215220225                                                                     SerTrpThrGlnAspArgMetTrpArgLysAsnArgSerArgAsn                                 230235240                                                                     GlnAsnSerThrCysIleGlyThrAspLeuAsnArgAsnPheAsp                                 245250255                                                                     ValSerTrpAspSerSerProAsnThrAsnLysProCysLeuAsn                                 260265270                                                                     ValTyrArgGlyProAlaProGluSerGluLysGluThrLysAla                                 275280285                                                                     ValThrAsnPheIleArgSerHisLeuAsnSerIleLysAlaTyr                                 290295300                                                                     IleThrPheHisSerTyrSerGlnMetLeuLeuIleProTyrGly                                 305310315                                                                     TyrThrPheLysLeuProProAsnHisGlnAspLeuLeuLysVal                                 320325330                                                                     AlaArgIleAlaThrAspAlaLeuSerThrArgTyrGluThrArg                                 335340345                                                                     TyrIleTyrGlyProIleAlaSerThrIleTyrLysThrSerGly                                 350355360                                                                     SerSerLeuAspTrpValTyrAspLeuGlyIleLysHisThrPhe                                 365370375                                                                     AlaPheGluLeuArgAspLysGlyLysSerGlyPheLeuLeuPro                                 380385390                                                                     GluSerArgIleLysProThrCysLysGluThrMetLeuSerVal                                 395400405                                                                     LysPheIleAlaLysTyrIleLeuLysAsnThrSer                                          410415417                                                                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 417 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetArgLeuThrLeuLeuLeuAlaAlaLeuLeuGlyTyrIleTyr                                 151015                                                                        CysGlnGluThrPheValGlyAspGlnValLeuGluIleIlePro                                 202530                                                                        SerHisGluGluGlnIleArgThrLeuLeuGlnLeuGluAlaGlu                                 354045                                                                        GluHisLeuGluLeuAspPheTrpLysSerProThrIleProGly                                 505560                                                                        GluThrValHisValArgValProPheAlaSerIleGlnAlaVal                                 657075                                                                        LysValPheLeuGluSerGlnGlyIleAspTyrSerIleMetIle                                 808590                                                                        GluAspValGlnValLeuLeuAspGlnGluArgGluGluMetLeu                                 95100105                                                                      PheAsnGlnGlnArgGluArgGlyGlyAsnPheAsnPheGluAla                                 110115120                                                                     TyrHisThrLeuGluGluIleTyrGlnGluMetAspAsnLeuVal                                 125130135                                                                     AlaGluAsnProGlyLeuValSerLysValAsnLeuGlySerSer                                 140145150                                                                     PheGluAsnArgProMetAsnValLeuLysPheSerThrGlyGly                                 155160165                                                                     AspLysProAlaIleTrpLeuAspAlaGlyIleHisAlaArgGlu                                 170175180                                                                     TrpValThrGlnAlaThrAlaLeuTrpThrAlaAsnLysIleAla                                 185190195                                                                     SerAspTyrGlyThrAspProAlaIleThrSerLeuLeuAsnThr                                 200205210                                                                     LeuAspIlePheLeuLeuProValThrAsnProAspGlyTyrVal                                 215220225                                                                     PheSerGlnThrThrAsnArgMetTrpArgLysThrArgSerLys                                 230235240                                                                     ArgSerGlySerGlyCysValGlyValAspProAsnArgAsnTrp                                 245250255                                                                     AspAlaAsnPheGlyGlyProGlyAlaSerSerSerProCysSer                                 260265270                                                                     AspSerTyrHisGlyProLysProAsnSerGluValGluValLys                                 275280285                                                                     SerIleValAspPheIleLysSerHisGlyLysValLysAlaPhe                                 290295300                                                                     IleThrLeuHisSerTyrSerGlnLeuLeuMetPheProTyrGly                                 305310315                                                                     TyrLysCysThrLysProAspAspPheAsnGluLeuAspGluVal                                 320325330                                                                     AlaGlnLysAlaAlaGlnAlaLeuLysArgLeuHisGlyThrSer                                 335340345                                                                     TyrLysValGlyProIleCysSerValIleTyrGlnAlaSerGly                                 350355360                                                                     GlySerIleAspTrpAlaTyrAspLeuGlyIleLysTyrSerPhe                                 365370375                                                                     AlaPheGluLeuArgAspThrAlaPheTyrGlyPheLeuLeuPro                                 380385390                                                                     AlaLysGlnIleLeuProThrAlaGluGluThrTrpLeuGlyLeu                                 395400405                                                                     LysThrIleMetGluHisValArgAspHisProTyr                                          410415417                                                                     __________________________________________________________________________

What is claimed is:
 1. A method for coagulating blood comprising addingto the blood an effective amount of an isolated monomeric human plasmacarboxypeptidase B (PCPB) having a molecular weight on non-reducingSDS-polyacrylamide gel electrophoresis of about 60 kD and having in itsmature form the N-terminal sequence PheGlnSer.